Yeast metabolism

Yeast Yeast 2005; 22 : 835–894. Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/yea.1249

Review

A history of research on yeasts 9: regulation of sugar metabolism1 James A. Barnett*† and Karl-Dieter Entian ‡ † School

of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK Mikrobiologie, Universität at Frankfurt, Marie-Curie-Straße 9, D-60439 Frankfurt/Main, Germany

‡ Institut f¨ ur ur

*Correspondence to: James A. Barnett, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK. E-mail: [email protected]

Keywords: Keywords: history history of yeast research; research; Pasteur effect; effect; Kluyver Kluyver effect; Custers Custers effect; Crabtree effect; glucose repression; glucose inactivation

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Pasteur effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pasteur’s observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Studies by Meyerhof, Warburg and others: 1920s and 1930s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-Phosphofructokinase: Engelhardt and Sakov . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Saccharomyces Saccharomyces cerevisiae cerevisiae and the Pasteur effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Custers effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Kluyver effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kluyver’s observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Observations of Sims and Barnett . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experiments of Jack Pronk and his colleagues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kluyver effect mutants: fds and gal2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Crabtree effect (repression of respiration) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucose repression in yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic analysis of glucose repression and identification of the genes involved . . . . . . . . . . . . . . . . . . Nomenclature of genes and their synonyms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Zimmermann’s selection system for mutants defective in glucose repression . . . . . . . . . . . . . . . . . . Entian’s analysis of hexokinases and their r ole oˆ le in glucose repression . . . . . . . . . . . . . . . . . . . . . . . . . Carlson’s analysis of sucrose-non-fermenting ( snf ) ) mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Repressors and activators under regulatory control of the Snf/Cat kinase . . . . . . . . . . . . . . . . . . . . . The current model of glucose repression: single and double control systems . . . . . . . . . . . . . . . . . . . . Classification of glucose-repressible genes according to their regulation . . . . . . . . . . . . . . . . . . . . . . Enzyme inactivation and the regulation of gluconeogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Holzer’s analyses of glucose inactivation (catabolite inactivation) . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic analysis of glucose inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucose inactivation: proteasomal versus vacuolar degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Previous articles in this series: [21–26,28,30,31].

Copyright © 2005 John Wiley & Sons, Ltd.

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J. A. Barnett and K.-D. Entian

Introduction The present article continues the description of the history of research on induction and repression of individual enzymes, begun in number 7 of this series [25]. Herein are described some quite recent findings, which were made after molecular biological methods had become usual for studying these regulatory processes in yeasts. Some account is also given below of the investigation of certain well-known, general regulatory mechanisms that control sugar metabolism, and which involve enzyme induction, repression and inactivation. These mechanisms which regulate sugar metabolism have been called the Pasteur, Kluyver, Custers and Crabtree effects (naming the scientists who first described the respective phenomena), glucose or catabolit catabolitee repression repression,, and glucose glucose or catabolit catabolitee inactivat inactivation ion (Table (Table 1). What has been called the Crabtree Crabtree effect effect in yeasts should should,, as will will be discus discussed sed below, below, be called called ‘gluco ‘glucose se repres repressio sion’. n’. Such Such regula regulator tory y effects effects involve involve enzyme enzyme synthesis and enzyme enzyme activity (Table (Table 2). 2). In describin describing g the original original findings findings and the development of later research, an attempt is made to give clear definitions of the phenomena cerevisiae and, descri described bed,, as well well as an exposi expositio tion n of their their physio physiolog logica icall rôles oles in Saccharomyces cerevisiae and, as far far as is know known, n, in othe otherr yeas yeasts ts too. too. An acco accoun untt is also also give given n of the the hist histor ory y of rese resear arch ch on the the 2 inter-regu inter-regulatio lation n of glycolysi glycolysiss and gluconeog gluconeogenesi enesis. s. The story of studying these processes, like many aspect aspectss of microb microbiol iology ogy,, began began with with the work of Louis Louis Pasteu Pasteurr 3 who, who, in 1861, 1861, descri described bed how the growth of yeast per gram of sugar consumed was much greater under aerobic than anaerobic conditions [281].

The Pasteur effect Many Many kinds kinds of cell cell utiliz utilizee exogen exogenous ouslyly-sup suppli plied ed sugar sugar faster faster under under anaero anaerobic bic than than under under aerobi aerobicc condit condition ions. s. This This is the Pasteur Pasteur effec effect. t. Howeve However, r, the term has been used used variou variously sly and the effect effect has been been report reported ed as occurr occurring ing in many many differ different ent organism organismss and tissues. tissues. There has been a great great deal deal of confused writing on the subject, as indicated in the three quotations from the 1930s which follow this paragr paragraph aph,, and the large large number numberss of public publicati ations ons on this this topic topic have have been been review reviewed ed extens extensive ively ly (e.g. (e.g. [46,83,84,203,226,293,329,336]). The intimate relations between the two processes [oxidation and fermentation] has occupied many biochemists since Pasteur discovered their quantitative interdependence, now known as the ‘Pasteur Reaction’. Pasteur found that there was some sort of equilibrium between oxidation and fermentation. If oxidation is suppressed by lack of oxygen, fermentation begins. If we promote again oxidation, fermentation is set to rest. The mechanism of this relation has been one of the most attractive puzzles of biochemistry ever since (Albert von Szent-Györgyi orgyi 1937 [365, p. 166]). By far the great majority [of experts on the Pasteur effect] . . . belong to a class which, vaguely aware of the Pasteur effect . . . rather accidentally obtain some sort of Pasteur effect, often with some special organism and set of conditions, conditions, and announce boldly, not infrequently infrequently in Nature (or in the good old days, Naturwissenschaften ), that here is the explanation of the Pasteur effect. It is this human, indeed lovable, but mathematically-impossible-thatthey-could-all-be-right class that we must be wary of (Dean Burk 4 1939 [46, p. 421]). 2

Glycolysis is the anaerobic breakdown of sugar to pyruvate; gluconeogenesis is the formation of D-glucose from compounds which are not carbohydrates. 3 Some accounts of the following scientists, who are mentioned here, are given in earlier articles of this series: C. F. Cori [23], E. Fischer [28], E. F. Gale [25], J. S. Haldane, D. Keilin, E. P. Kennedy [24], A. J. Kluyver [30], H. A. Krebs [23], A. L. Lehninger [31], F. A. Lipmann [23], B. Magasanik [25], O. F. Meyerhof [23], J. Monod [25], P. Ostern [23], L. Pasteur [22], A. Sols [23], S. Spiegelman [25], A. von Szent-Gy¨ Szent-Gyorgyi o¨ rgyi [24], O. Warburg [23]. 4 American biochemist, biochemist, worked at University University College London, the Kaiser Wilhelm Wilhelm Institute in Berlin and Dean Burk (1904–1988), American Harvard and Cornell Universities. He became chief chemist at the National Cancer Institute, Bethesda [8].


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Introduction The present article continues the description of the history of research on induction and repression of individual enzymes, begun in number 7 of this series [25]. Herein are described some quite recent findings, which were made after molecular biological methods had become usual for studying these regulatory processes in yeasts. Some account is also given below of the investigation of certain well-known, general regulatory mechanisms that control sugar metabolism, and which involve enzyme induction, repression and inactivation. These mechanisms which regulate sugar metabolism have been called the Pasteur, Kluyver, Custers and Crabtree effects (naming the scientists who first described the respective phenomena), glucose or catabolit catabolitee repression repression,, and glucose glucose or catabolit catabolitee inactivat inactivation ion (Table (Table 1). What has been called the Crabtree Crabtree effect effect in yeasts should should,, as will will be discus discussed sed below, below, be called called ‘gluco ‘glucose se repres repressio sion’. n’. Such Such regula regulator tory y effects effects involve involve enzyme enzyme synthesis and enzyme enzyme activity (Table (Table 2). 2). In describin describing g the original original findings findings and the development of later research, an attempt is made to give clear definitions of the phenomena cerevisiae and, descri described bed,, as well well as an exposi expositio tion n of their their physio physiolog logica icall rôles oles in Saccharomyces cerevisiae and, as far far as is know known, n, in othe otherr yeas yeasts ts too. too. An acco accoun untt is also also give given n of the the hist histor ory y of rese resear arch ch on the the 2 inter-regu inter-regulatio lation n of glycolysi glycolysiss and gluconeog gluconeogenesi enesis. s. The story of studying these processes, like many aspect aspectss of microb microbiol iology ogy,, began began with with the work of Louis Louis Pasteu Pasteurr 3 who, who, in 1861, 1861, descri described bed how the growth of yeast per gram of sugar consumed was much greater under aerobic than anaerobic conditions [281].

The Pasteur effect Many Many kinds kinds of cell cell utiliz utilizee exogen exogenous ouslyly-sup suppli plied ed sugar sugar faster faster under under anaero anaerobic bic than than under under aerobi aerobicc condit condition ions. s. This This is the Pasteur Pasteur effec effect. t. Howeve However, r, the term has been used used variou variously sly and the effect effect has been been report reported ed as occurr occurring ing in many many differ different ent organism organismss and tissues. tissues. There has been a great great deal deal of confused writing on the subject, as indicated in the three quotations from the 1930s which follow this paragr paragraph aph,, and the large large number numberss of public publicati ations ons on this this topic topic have have been been review reviewed ed extens extensive ively ly (e.g. (e.g. [46,83,84,203,226,293,329,336]). The intimate relations between the two processes [oxidation and fermentation] has occupied many biochemists since Pasteur discovered their quantitative interdependence, now known as the ‘Pasteur Reaction’. Pasteur found that there was some sort of equilibrium between oxidation and fermentation. If oxidation is suppressed by lack of oxygen, fermentation begins. If we promote again oxidation, fermentation is set to rest. The mechanism of this relation has been one of the most attractive puzzles of biochemistry ever since (Albert von Szent-Györgyi orgyi 1937 [365, p. 166]). By far the great majority [of experts on the Pasteur effect] . . . belong to a class which, vaguely aware of the Pasteur effect . . . rather accidentally obtain some sort of Pasteur effect, often with some special organism and set of conditions, conditions, and announce boldly, not infrequently infrequently in Nature (or in the good old days, Naturwissenschaften ), that here is the explanation of the Pasteur effect. It is this human, indeed lovable, but mathematically-impossible-thatthey-could-all-be-right class that we must be wary of (Dean Burk 4 1939 [46, p. 421]). 2

Glycolysis is the anaerobic breakdown of sugar to pyruvate; gluconeogenesis is the formation of D-glucose from compounds which are not carbohydrates. 3 Some accounts of the following scientists, who are mentioned here, are given in earlier articles of this series: C. F. Cori [23], E. Fischer [28], E. F. Gale [25], J. S. Haldane, D. Keilin, E. P. Kennedy [24], A. J. Kluyver [30], H. A. Krebs [23], A. L. Lehninger [31], F. A. Lipmann [23], B. Magasanik [25], O. F. Meyerhof [23], J. Monod [25], P. Ostern [23], L. Pasteur [22], A. Sols [23], S. Spiegelman [25], A. von Szent-Gy¨ Szent-Gyorgyi o¨ rgyi [24], O. Warburg [23]. 4 American biochemist, biochemist, worked at University University College London, the Kaiser Wilhelm Wilhelm Institute in Berlin and Dean Burk (1904–1988), American Harvard and Cornell Universities. He became chief chemist at the National Cancer Institute, Bethesda [8].


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Table 1. Regulatory phenomena Name

What happens

Underlying factors

Some key references

PASTEUR EFFECT

Sugar used faster anaerobically than aerobically (insignificant in Saccharomyces cerevisiae)

Oxidized cytochrome inactivates 6-phosphofructokinase

Pasteur 1861 [282]; Meyerhof 1925 [258]; Warburg 1926 [367]; Lipmann 1933, 1934 [224,225]; Engelhardt and Sakov 1943 [95]; Lagunas and Gancedo [211]

Brettanomyces and Dekkera spp. ferment D-glucose to ethanol and CO2 faster in aerobic than in anaerobic conditions

Much acetic acid is produced via an NAD+ -aldehyde dehydrogenase. Consequently, anaerobically, the high high NADH: NAD+ ratio inhibits glycolysis

´ ´ and Custers 1940 [73], Wik en colleagues 1961 [382], Scheffers 1961, 1966 [312,313]

K LUYVER LUYVER EFFECT

Ability to use oligosaccharide or galactose aerobically, but not anaerobically, although glucose is fermented

Probably caused mainly by slower uptake of sugar anaerobically

Kluyver and Custers 1940 [194], Sims and Barnett 1978 [324], Barnett Barnett and Sims 1982 [32], Barnett 1992 [20], Weusthuis and colleagues 1994 [378,379]

CRABTREE EFFECT

Adding glucose to tumour cells lowers the respiration rate

Decrease of ADP concentration in mitochondria

Crabtree 1929 [71], Ibsen 1961 [175]

GLUCOSE

Repr Repres essi sion on of resp respir irat atio ion n

Repr Repres essi sion on of stru struct ctur ural al gene geness of respiratory enzymes

Spiegelman and Reiner 1947 [331], Magasanik 1961 [240], Bartley and colleagues colleagues [287 –289], Zimmermann and colleagues 1977 [398,399], Entian and Mecke 1982 [106], Nehlin and colleagues 1991 [269], DeVit and colleagues 1997 [82], Gancedo 1998 [129], Carlson 1999 [47]

Decrease of enzyme activity within minutes after adding glucose

Phosphorylation (rapidly reversible) and proteolytic degradation (irreversible) of enzyme

Holzer and colleagues 1966 [390], Gancedo 1971 [125], Lenz and Holzer 1980 [220], Entian and colleagues 1983 [102], Rose and colleagues 1988 [306], Hämmerle ammerle and colleagues 1998 [152], Sch ule u¨ le and colleagues 2000 [318]

CUSTERS EFFECT

REPRESSION

(glucose effect, carbon catabolite repression)

GLUCOSE INACTIVATION

(catabolite inactivation)

As considerable confusion exists in the current literature as to the real nature of the Pasteur effect, it is necessary to explai explain n Pasteu Pasteur’s r’s origin original al concep concepti tions ons and to descri describe be his experi experimen mental tal results results on the effect effect of oxygen oxygen on carbohydrate catabolism (Kendal Dixon5 1937 [84, p. 432]).

Pasteur’s observations The first relevant publication was that of Pasteur himself, who in 1861 found the growth yield of brewer’s yeast, per gram of sugar consumed, to be many times greater aerobically than anaerobically. Eventually, this observation was shown to have very wide significance for understanding the biochemistry of many kinds of cell which are capable of both aerobic and anaerobic metabolism. Pasteur put 100 cm 3 of sugar 5

Kendal Cartwright Dixon (1911–1990), Irish biochemist and medical man, worked on carbohydrate and lipid metabolism in Cambridge from 1933, where he became Professor of Cellular Pathology [9].


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Table 2. Major regulatory mechanisms in carbohydrate metabolism Kind of regulation

Physiological observation

Examples

Mechanism

Mechanisms regulating enzymic activity Allosteric activation and inactivation

Immediate reversible gain or loss of enzymic activity

6-Phosphofructokinase pyruvate kinase

Activators or inhibitors change substrate affinity

Interconversion by covalent modification

Reversible loss of enzymic activity within minutes

Fructose-1,6-bisphosphatase

Usually phosphorylation of enzyme

Inactivation

Irreversible loss of enzymic activity

Fructose-1,6-bisphosphatase and other mainly gluconeogenic and glyoxylate cycle enzymes

Specific proteolysis of the enzyme

Mechanisms regulating enzyme synthesis Induction

Increase in enzymic activity in response to presence of inducer (substrate or structurally similar compound)

GAL and MAL genes

Activation of transcription upon binding of specific gene activators

Repression

No further enzyme synthesis due to a stop of transcription of the encoding gene

Genes encoding glucose-repressible enzymes

Inhibition of transcription upon binding of specific gene repressors

Derepression

Increase in specific activity of enzyme after removing repressing substrate

Genes encoding glucoserepressible enzymes

Release from repression due to de-binding of gene repressors

solution with a little protein into a 250 cm 3 flask and boiled the solution to remove the oxygen. After cooling, he introduced a very small amount of beer yeast and placed the drawn-out neck of the flask under mercury (see [22]). The yeast grew only a little and the sugar was fermented: 60 to 80 parts of sugar were consumed for 1 part of yeast formed. He wrote: If the experiment is done in contact with the air and over a large surface area . . . much more yeast is produced for the same quantity of sugar consumed. The air loses oxygen which is absorbed by the yeast. The latter grows vigorously, but its characteristic capacity to ferment tends to disappear in these conditions. For one part of yeast formed, only 4 to 10 parts of sugar are transformed. The yeast nevertheless retains its capacity to cause fermentation. Indeed fermentation appears greatly increased if the yeast is again cultured with sugar in the absence of free oxygen.6

Studies by Meyerhof, Warburg and others: 1920s and 1930s As a sequel to Pasteur’s observations, in the 1920s Otto Meyerhof and Otto Warburg examined differences between the aerobic and anaerobic breakdown of sugar in yeast, muscle and other tissues. Various tissues, such as muscle, were already known to form lactate from sugar in the absence of oxygen (see [23]). Wanting to test whether oxygen uptake increases when cells begin to grow [366], Warburg compared the respiration rates of certain rat cancer cells with those of normal rat cells [372,376]. He found the cancer cells to have (a) the same rate of oxygen consumption as the normal cells but (b) a much higher 6

Si l’expérience est faite au contact de l’air et sur une grande surface. . .Pour la même quantité de sucre disparu, il se fait beaucoup plus de levûre. L’air en contact cède de l’oxygène qui est absorbé par la levûre. Celle-ci se développe e´ nergiquement, mais son caractère de ferment tend a` disparaˆıtre dans ces conditions. On trouve en effet que pour 1 partie de levûre formé e, il n’y aura que 4 a` 10 parties de sucre transformé . Le rôle de ferment de cette levûre subsiste néanmoins et se montre même fort exalté si l’on vient a` la faire agir sur le sucre en dehors de l’influence du gaz oxyg` ene libre [282, p. 80].


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rate of lactate formation, even in the presence of oxygen. In addition, ethyl isocyanide, which inhibited heavy-metal catalysis of certain oxidations, abolished the slowing down of glycolysis by oxygen. From such observations, he concluded: Respiration and fermentation are thus connected by a chemical reaction, which I call the ‘Pasteur reaction’ after its discoverer.7

Working with both yeast and muscle, it was Meyerhof who was the first to examine Pasteur’s observations of differences between the aerobic and anaerobic breakdown of carbohydrate. Meyerhof found that glycogen was catabolized by frog muscle more slowly when in oxygen than in nitrogen [257]. Then, working with several kinds of yeast, he showed indisputably that the rate of sugar breakdown by some yeasts is greater in the absence than in the presence of air [258]. He measured oxygen uptake and carbon dioxide output, using Warburg manometers, 8 and estimated the quotients Q O2 and QCO2 ,9 both with washed yeast at 25 ◦ C in phosphate solution ( 0.1 M KH2 PO4 ) and with a high concentration of D-glucose (∼0.28 M). A brewer’s bottom yeast had about the same rate of oxygen uptake, whether in buffer alone or when supplied with glucose, and the high rate of carbon dioxide production was similar (QCO2 > 200 ) in air or under nitrogen (Table VA of [258]). This finding of Meyerhof’s has since been reported many times for strains of Saccharomyces cerevisiae (e.g. [342]): that is to say, with a high concentration of glucose, sugar catabolism is entirely anaerobic, even in aerated cultures. Hence, for such a yeast, the Pasteur effect cannot occur. Warburg had already found that carbon monoxide inhibits the respiration of baker’s yeast by combining with a component of the respiratory system of the cell [369]. During a visit to Warburg in the winter of 1927– 1928, the English physiologist Archibald Hill 10 told him about the light-sensitivity of the carbon monoxide–haemoglobin complex discovered in 1896 by John Scott Haldane and James Smith 11 [151], [204, p. 26]. Promptly investigating, Warburg found that the carbon monoxide compound of his ‘respiratory enzyme’ ( Atmungsferment , see [24]), was also light-sensitive (Figure 1). So, by illuminating his yeast suspensions with monochromatic light of different wavelengths and known intensities, he measured the absorption spectrum of this Atmungsferment [373– 375]. Furthermore, measuring the inhibition of respiration by his yeast in different mixtures of carbon monoxide and oxygen (replacing carbon monoxide by nitrogen as a control), Warburg was able to calculate the relative affinity ( K ) of his Atmungsferment for oxygen and carbon monoxide as a partition constant: K =

n

.

[CO]

(1 − n ) [O2 ]

where n is the ratio of the respiration rate in the presence of carbon monoxide to the rate in its absence. Hans Krebs comments that: . . . to devise and to carry out the experiments and to develop the mathematical analysis of the measurements required very exceptional experimental and theoretical skill. First he [Warburg] had to find sources of monochromatic light of sufficient intensity, then he needed methods for measuring the gas exchanges and light intensities, and finally he had to elaborate the theory for the quantitative interpretation of the measurements . . . It was this work for which Warburg was awarded the Nobel Prize for Medicine and Physiology in 1931 [204, p. 27]. 7

Atmung und Gärung sind also durch eine chemische Reaktion verbunden, die ich nach ihrem Entdecker ‘Pasteur sche Reaktion’ nenne [367, p. 435]. 8 Warburg manometers are described in article 5 of this series [23, p. 516]. 9 QO2 and QCO2 were expressed as mm3 of O2 taken up or of CO2 produced, respectively, per mg dry weight of yeast per hour. 10 Archibald Vivian Hill (1886– 1977), English physiologist, was professor first at Manchester University from 1923, then at University College London from 1926. He shared the 1922 Nobel Prize for physiology or medicine with Otto Meyerhof for work on heat production in muscle contraction [187]. 11 James Lorrain Smith (1862–1931), Scottish physiologist, worked at Oxford with J. S. Haldane on air pollution caused by breathing. He moved to Queen’s College, Belfast, in 1894, where he became professor in 1901. Subsequently he held chairs in Manchester and Edinburgh [150].


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Figure 1. Results of one of Warburg’s experiments on the action of light on the carbon monoxide inhibition of yeast respiration. Reproduced with permission from [371, p. 81]. Dunkel = dark; hell = light

The Pasteur effect was studied further in many other kinds of cell. Using Warburg’s methods to investigate the effects of carbon monoxide on the aerobic metabolism of several animal tissues, Hans Laser12 confirmed some of Warburg’s observations, which are shown in Figure 1, and found the following: (a) the rate of respiration was the same in oxygen + carbon monoxide as in oxygen + nitrogen mixtures; (b) replacing nitrogen by carbon monoxide increased the rate of glycolysis to that in fully anaerobic conditions; (c) the effect of carbon monoxide was reversed by light and he showed that, whereas respiration was unaffected, aerobic glycolysis increased up to the level of anaerobic glycolysis [213].

6-Phosphofructokinase: Engelhardt and Sakov In 1933, Fritz Lipmann suggested that the Pasteur effect might be a consequence of the oxidation of a glycolytic enzyme by an electron carrier, such as a cytochrome [224]. As a development of Lipmann’s view, in 1943, Vladimir Engelhardt 13 and Nikolai Sakov14 established the major rô le of 6-phosphofructokinase15 in producing the Pasteur effect [95]. Using fractionated muscle extract, they investigated sensitivity to oxidation (by various redox dyes 16 ) of certain enzymes of the glycolytic 12

Hans Laser (1899–1980), German biochemist, worked at the Kaiser Wilhelm Institute for Cell Physiology at Berlin, but came to England as a refugee from the Nazi government in 1934. He worked for over 30 years at the Molteno Institute, Cambridge, where his research included a study of lysis of cells in patients with malaria and the study of neoplastic cells [7]. 13 Vladimir Alexandrovich Engelhardt (1894–1984) was a great and much-liked Russian biochemist, who discovered oxidative phosphorylation and the functioning of myosin as an ATPase. He was professor of biochemistry at Kazan University from 1929 and from 1935 at the Institute of Biochemistry of the Academy of Sciences of the USSR in Moscow whence, in the early 1940s when the war was approaching Moscow, he was evacuated to Kazakhstan in Central Asia [94,192,326]. 14 Nikolai E. Sakov died in the battle for Stalingrad in 1942, his joint work with Engelhardt having been completed in 1941 [94,192]. 15 6-Phosphofructokinase was discovered in 1936 by Pawel Ostern and his colleagues [275], see [23]. 16 Redox dyes are mostly coloured when oxidized and colourless when reduced. Engelhardt and Sakov found inhibition by dyes with E  0 > + 0.05 V, such as 2,6-dibromophenolindophenol or 2,6-dichlorophenolindophenol. E  0 is the approximate electrode potential, when there are equal concentrations of both oxidized and reduced forms at pH 7. Relations of the oxidation–reduction (‘redox’) potential, electromotive force and ionic concentration had been worked out in 1889 by Hermann Walther Nernst (1864–1941) [272].


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pathway and found only one of them to be sensitive, namely, 6-phosphofructokinase (PFK), which catalyses the following reaction: D-fructose

6-phosphate + ATP −−→ D-fructose 1,6-bisphosphate

They also showed that oxidized cytochrome inactivated PFK, as Engelhardt relates: Evidently, the effect of these agents, completely alien to the normal catalytic system of the cell, even if highly suggestive, was only of an indirect kind. But an impressive proof of the validity of the findings was obtained when an exactly similar effect was found using the major physiological oxidizing system, cytochrome and its oxidase. In the presence of a suitable intermediate carrier, oxidized cytochrome by itself taken in stoichiometric amount, inhibited the phosphofructokinase. But, most important, the inhibition could be obtained with minute, catalytic amounts of cytochrome in the presence of cytochrome oxidase. In air, almost complete inhibition is observed, whereas in nitrogen no inhibition occurs. This experiment can well be regarded as the closest modelling of the Pasteur effect under the most simplified conditions [94, pp. 9–10].

Since this work was finished during the middle of World War II, it was impracticable for the Russian authors to send their script abroad, so it was published in Russian in the journal Biokhimia. Consequently, as it was not widely known, this work was not cited in the 1950s and early 1960s by the various authors who presented evidence that changes in PFK activity underlie the Pasteur effect (e.g. [279,309]). At the same time as these Russian experiments, two American workers were obtaining results consistent with those of Engelhardt and Sakov. First, Carl Cori was suggesting that PFK has a regulatory r oˆ le in muscle glycolysis, writing: . . . hexosemonophosphate . . . a normal constituent of muscle . . . can increase considerably under certain experimental conditions without any increase in the formation of lactic acid. This indicates that the reaction between fructose-6-phosphate and adenosinetriphosphate in intact muscle is a limiting factor as regards the rate at which lactic acid is formed and carbohydrate is oxidized [70, p. 183].

Second, Joseph Melnick’s 17 findings, published in 1941 and 1942, accorded with the suggestion that the Pasteur effect could be brought about by the action of cytochrome and cytochrome oxidase on PFK. The photochemical absorption spectra, obtained with baker’s yeast, indicated that the three proteins, known as (a) ‘Pasteur enzyme’, (b) Atmungsferment or (c) cytochrome oxidase, were all the same enzyme [253,254,333]. As David Keilin wrote: It is, therefore, reasonable to assume that cytochrome oxidase is the component showing the light-sensitive inhibition by carbon monoxide and the photochemical absorption spectrum of the catalytic system involved in the Pasteur reaction [188, p. 268].

The allosteric effectors of 6-phosphofructokinase have been identified relatively recently, and the effect of their inhibition is different for various organisms. Many allosteric inhibitors (more than 20, including cytochrome) for 6-phosphofructokinases have been found in vitro . However, in vivo , the major allosteric inhibitor of 6-phosphofructokinase is ATP and the major allosteric activators are fructose 2,6-bisphosphate and AMP. The extent of activation and inhibition by these effectors differs between organisms. Fructose 2,6-bisphosphate, first discovered in mammalian cells [357], is the main activator of 6-phosphofructokinase18 in S. cerevisiae (see Figure 19, below) [191], (for review see [40]). 17

Joseph Lewis Melnick (1914–2001), American medical microbiologist, worked especially on enteroviruses at Yale University. He became Professor of epidemiology in 1954 (Historical Register of Yale University, 1951–1968, p. 523) and moved to a chair at Baylor College of Medicine, Houston, in 1958 [38,255,364]. 18 S. cerevisiae has two enzymes that phosphorylate D-fructose 6-phosphate. The best known glycolytic enzyme, named 6phosphofructokinase-1, is a heterooctamer with 4 α - and 4 β -subunits [195], which are encoded by genes PFK1 (α -subunit) and PFK2 (β -subunit) [67,159]. 6-Phosphofructokinase-1 phosphorylates D-fructose 6-phosphate to the glycolytic intermediate fructose 1,6bisphosphate, whereas 6-phosphofructokinase-2 (encoded by PFK26 and PFK27 ) phosphorylates D-fructose 6-phosphate to D-fructose 2,6-bisphosphate.


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Table 3. The Pasteur effect: chronology of some findings Date

Author

Findings

1861

Pasteur [281,282]

Brewer’s yeast growth yield per gram of sugar used is 20 times greater aerobically than anaerobically

1892

Brown [42]

With high concentration of brew er’s top yeast (so growth w as insignificant), fermentation found to be independent of oxygen supply

1920

Meyerhof [257]

Rate of glycogen breakdown in muscle greater anaerobically than aerobically

1925

Meyerhof [258]

Rate of sugar breakdown by some yeasts greater anaerobically than aerobically

1926

Warburg [367]

1. The term Pasteursche Reaktion used for a hypothetical chemical reaction linking respiration and fermentation. 2. CO inhibits yeast respiration.

1928

Warburg and Negelein [373–375]

CO + ‘respiratory enzyme’ complex cleaved by light and absorption spectrum of enzyme determined

1933

Lipmann [224]

Oxidizing agents stop glycolysis in yeast and muscle: O 2 itself inhibits glycolysis

1937

L aser [213]

In several animal tissues, CO increases glycolysis but does not alter respiration rate

1941

Stern and Melnick [253,333]

Light reverses formation of CO-enzyme complex in yeast and retina

1943

Engelhardt and Sakov [95]

Cytochrome + cytochrome oxidase inactivated 6-phosphofructokinase

1962

Passoneau and Lowry [279]

‘Rediscovery’ of Engelhardt and Sakov’s findings

1980

Van Schaftingen, Hue and Hers [357]

Fructose 2,6-bisphosphate stimulates 6-phosphofructokinase

Table 3 shows the chronological sequence of some of the work on the Pasteur effect.

Saccharomyces cerevisiae and the Pasteur effect In 1966 R. H. De Deken 19 recorded differences between a number of yeast species in their rates of oxidative respiration and of non-oxidative fermentation, when growing aerobically on D-glucose [78]. The yeasts varied from those that are completely oxidative under these conditions, such as Candida utilis, to others that are completely fermentative, such as Schizosaccharomyces pombe (Table 4). The figures given in the table are simply illustrative, since considerable variations occur between strains of the same species and under differing experimental conditions. However, De Deken’s results indicate clearly that C. utilis [227] and Kluyveromyces lactis [308] are both likely to be better yeasts for studying the Pasteur effect than Saccharomyces cerevisiae . The occurrence of the Pasteur effect has also been described in Saccharomyces bayanus (uvarum ), Schizosaccharomyces pombe [227] and Schwanniomyces occidentalis [286]. But there are special problems in interpreting much of the work published on the Pasteur effect in yeasts. Because Pasteur’s original observations were on yeast, and to biochemists ‘yeast’ usually means Saccharomyces cerevisiae, most of the work has been done with that species. Now, as indicated by 19

R. H. De Deken (1927–?) worked on yeast biochemistry and biochemical cytology at the Institut de Recherches du Centre d’Enseignement et de Recherches des Industries Alimentaires et Chimiques (Brussels) in the 1950s and 1960s.


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Table 4. Rates of oxidative respiration and non-oxidative fermentation by several yeasts growing aerobically in 17 mM-D-glucose at pH 6.5 (temperature and growth phase unspecified). Rates are in µ l of gas per 107 yeast cells per 10 min at atmospheric pressure (results of De Deken 1966 [78]). Names in parentheses are those given by De Deken but are not in use currently Yeast

Oxygen uptake

Carbon dioxide evolved by fermentation

Saccharomyces cerevisiae (italicus)

0.0

94.5

Kluyveromyces thermotolerans (Torulopsis dattila)

0.0

52.0

Schizosaccharomyces pombe

0.0

40.6

Dekkera bruxellensis (Brettanomyces lambicus)

1.2

9.3

Torulaspora delbrueckii (Torulopsis colliculosa)

10.7

30.2

Kluyveromyces lactis (Torulopsis sphaerica)

25.7

3.5

Candida tropicalis

27.7

0.9

Pichia (Hansenula) anomala

24.1

0.0

Candida utilis

30.0

0.0

De Deken’s observations, as well as by some of Meyerhof’s results described above, D-glucose almost completely represses the aerobic metabolism of many strains of S. cerevisiae, even when oxygen is present . Accordingly, such yeasts in the presence of glucose cannot show the Pasteur effect. Indeed, Rosario Lagunas, studying two strains, found the Pasteur effect to be insignificant during growth on glucose, galactose or maltose and very low during ammonia starvation [208]. Furthermore, Walter Bartley 20 (Figure 2) and his colleagues stated that cells of S. cerevisiae grown on glucose (at 50 m M or more) do not form mitochondria [289], the enzymes of the tricarboxylic acid cycle being repressed [287]. However, detecting mitochondria 21 in anaerobically grown or glucose-repressed S. cerevisiae requires special techniques for fixing and staining [72]. Since the 1960s, it has been accepted that this yeast when metabolizing anaerobically does have mitochondria in a smaller, somewhat elusive form [74] and these have sometimes been called ‘promitochondria’ [285,311]. In the 1970s, Barbara Stevens, by means of a remarkable electron micrographic study of serial thin sections and computer-aided three-dimensional reconstructions, showed the volume of the ‘promitochondria’ to occupy about 3% of the cell volume in glucose-repressed cells, and as much as 10–12% in derepressed respiring cells [334]. Nonetheless, Lagunas and her colleagues have observed the Pasteur effect in Saccharomyces cerevisiae in resting (non-growing) cells [210,211], the resting condition being obtained by depriving the yeast of a source of nitrogen. When growing, the cells respired only 3–20% of the sugar they catabolized; whereas resting cells respired as much as 25–100%. Accordingly, it became practicable to detect the Pasteur effect in such resting cells. Lagunas and her colleagues attributed this reduced rate of fermentation ( >10% of that in growing cells) to inactivation of the transport system by which the sugar enters the cells [210]. Furthermore, they pointed out that previous studies of the Pasteur effect in Saccharomyces cerevisiae [169,237,258,309] had indeed been done with resting cells. Lagunas and Carlos Gancedo found, even 20

Walter Bartley (1916–1994), English biochemist, worked in Hans Krebs’ laboratory, first in Sheffield and then in Oxford as a technician and later as a research student. Bartley became deputy director of Krebs’ Medical Research Council Unit for Cell Metabolism at Oxford and returned to Sheffield in 1963 as professor of biochemistry [10,13,15]. 21 Mitochondria, the sites in eukaryotes of tricarboxylic acid cycle reactions and oxidative phosphorylation (see [24]).


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Figure 2. Walter Bartley. Photo courtesy of Joan Brown

for resting cells, ‘that the magnitude of the Pasteur effect is very small in S. cerevisiae ’ [211], Lagunas commenting further: S. cerevisiae shows physiological characteristics very different from those often reported even in good textbooks of microbiology and biochemistry. The fact that the yeast obtains a small benefit from aerobiosis and that [the] Pasteur effect is neither important nor was discovered in this microorganism should not be ignored any longer [209, p. 227].

To sum up, Pasteur’s finding is undoubtedly correct, namely, that the increase in cell mass anaerobically is much smaller than aerobically. However, what is now called ‘the Pasteur effect’ — the generalization that the presence of oxygen decreases the rate of sugar breakdown — does not occur in all yeasts, let alone all other organisms. Indeed, the Pasteur effect is insignificant in his own experimental organism, which was likely to have been Saccharomyces cerevisiae or S. pastorianus . Two characteristics of these particular yeasts may explain his findings. First, the lower growth yield anaerobically was probably because these yeasts are unable to synthesize ergosterol and unsaturated fatty acids in the absence of oxygen, as Arthur Andreasen 22 and Theodore Stier23 found in the 1950s [4,5]. Second, the biphasic (or diauxic) growth of S. cerevisiae on glucose 22

Arthur A. Andreasen , who worked with Stier at Bloomington, was with Lynferd Wickerham in the early 1940s at the University of Illinois, Urbana, working on preserving yeasts by freeze-drying for the degree of Master of Science [380]. 23 Theodore James Blanchard Stier (1903–1991), American cellular physiologist, was professor of physiology at Indiana University from 1947 (information kindly supplied by Kristen Walker of Indiana University Archives).


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Figure 3. Typical biphasic (diauxic) growth of Saccharomyces cerevisiae on D-glucose in aerobic batch culture. The first phase (about 0–6 h) is characterized by production of ethanol which, after the disappearance of glucose, is used as the carbon source for growth (from [186]). Reprinted from Advances in Applied Microbiology 28, G. Käppeli, Regulation of carbon metabolism in Saccharomyces cerevisiae and related yeasts: 181–209, copyright 1986, with permission from Elsevier

(Figure 3) may be the underlying reason for the higher yield of biomass when oxygen is present. In phase 1, glucose is fermented to ethanol; and in phase 2, the ethanol is respired. The change in free energy for the anaerobic conversion of D-glucose into ethanol, given by: C6 H12 O6 −−→ 2EtOH + 2CO2 ,

G  = −235 kJ[205]

is much less than that for the aerobic oxidation of D-glucose, given by: G  = −2873 kJ[205]

C6 H12 O6 + 6O2 −−→ 6CO 2 + 6H2 O,

so, when there is a change from anaerobic to aerobic conditions, less glucose is consumed. For S. cerevisiae and other fermentative yeasts, the rapid fermentative catabolism of glucose to ethanol, accompanied by secretion of acids, such as succinate (as Pasteur found in 1860 [280]) and acetate (reviewed in [277]), generates an environment in which yeasts have an advantage, as they are generally more acid- and ethanol-tolerant than most bacteria. Hence, where there are high concentrations of sugar, such as in rotting figs or grapes, these relatively slow-growing eukaryotic microbes can compete successfully with most (fast-growing) prokaryotes.

The Custers effect In 1940, when working in Albert Kluyver’s (Figure 4) laboratory in Delft, Mathieu Custers 24 studied yeasts of the genera Dekkera and Brettanomyces, which are important in the brewing of the rather acid Belgian lambic beer [147]. In contrast to the Pasteur effect, Custers described how these yeasts ferment D-glucose to ethanol faster under aerobic conditions than anaerobically [73]. He also reported that they produce considerable amounts of acetic acid in addition to the ethanol. Custers called this behaviour of Brettanomyces the ‘negative Pasteur effect’ (see [382]). Lex Scheffers and his colleagues confirmed the 24

Mathieu Theodoor Jozef Custers , Dutch microbiologist, defended his doctor’s thesis on 3 May 1940, 1 week before the German army invaded The Netherlands. He became a school teacher in Amsterdam and died before 1970 (W. A. Scheffers, personal communication).


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Figure 4. Albert Jan Kluyver. Photo courtesy of C. T. Kluyver

existence of this effect in a number of strains of Brettanomyces and Dekkera [382] and renamed it the ‘Custers effect’ in 1966 [313]. Measuring respiratory exchanges with Warburg manometers, Scheffers found a marked Custers effect in Dekkera anomala ( Brettanomyces claussenii ), which was harvested from shaken aerobic cultures [312]. He also reported the stimulation in D. anomala of ‘anaerobic fermentation’ by various additions to the suspensions of this yeast. These additives included acetone, ether, acetaldehyde, acetone, pyruvic acid, formaldehyde, 3-hydroxy-2-butanone (acetoin 25 ), 1,3-dihydroxyacetone, butanone (methyl ethyl ketone) and α-oxoglutaric acid. He wrote: The results suggest an action of the carbonyl compounds as H-acceptors in enzymatic dehydrogenation . . . Oxidized coenzyme I (DPN) [NAD+ ] enhances anaerobic fermentation to an extent depending on its concentration . . . it is tentatively suggested that the inhibition of the start of fermentation in Br. claussenii under anaerobic conditions is, at least in part, due to a shortage of DPN. This inhibition is abolished on addition of O2 or of other substances able to oxidize DPNH enzymatically [312, p. 41].

Later, Scheffers described how, on adding exogenously the hydrogen-acceptor, 3-hydroxy-2-butanone, the rate of fermentation by Dekkera bruxellensis ( Brettanomyces intermedius ) is increased when under anaerobic conditions (Figure 5) [313]. He and his colleagues published additional evidence that glycolysis is slowed by lowering the concentration of NAD + (Figure 6) [50]. This is because production of acetic acid involves reduction 26 of NAD+ . The NAD+ is restored by any system which re-oxidizes NADH, such as NADH dehydrogenase, an electron carrier of the respiratory chain. 25

Acetoin (3-hydroxy-2-butanone) may be reduced to butane-2,3-diol by the action of butanediol dehydrogenase: CH3 · CO · CH(OH) · CH3 + NADH + H+ → CH 3 · CH(OH) · CH(OH) · CH3 + NAD+ [79]. 26 As described for bacterial acetate production, such as by Pseudomonas fluorescens [178].


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Figure 5. Fermentation of D-glucose by Dekkera bruxellensis (CBS 1943). Results of Scheffers, published in 1966. Reproduced from [313], courtesy W. A. Scheffers and by permission of Nature Publishing Group. Symbols: ž, in aerobic conditions + or − exogenous 10−3 M 3-hydroxy-2-butanone (acetoin); , in anaerobic conditions;  , in anaerobic conditions + 10−3 M 3-hydroxy-2-butanone; , with 0.12% oxygen

°

Figure 6. Custers effect: reduction of NAD(P)+ by formation of acetate from acetaldehyde lowers the concentration of NAD+ , which is necessary for oxidizing glyceraldehyde 3-phosphate in glycolysis

The Kluyver effect

Kluyver’s observations In 1940, Kluyver and Custers reported that although Candida (Torulopsis ) utilis can ferment D-glucose anaerobically to ethanol and carbon dioxide, this yeast can (unlike Saccharomyces cerevisiae ) utilize maltose aerobically only. Thus they confirmed earlier reports that certain yeasts were able to use the Copyright © 2005 John Wiley & Sons, Ltd.

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Table 5. Abilities of Candida utilis and Saccharomyces cerevisiae to utilize D-glucose and maltose

D-glucose

maltose

  aerobic growth  fermentation   aerobic growth  fermentation

Candida utilis

Saccharomyces cerevisiae

+

+

+

+

+

+

−

+

component hexoses of certain disaccharides anaerobically , yet could use those disaccharides aerobically only [194]. Thirty-eight years later, this phenomenon was named the Kluyver effect [324]. The problem of the Kluyver effect can be seen from Table 5. Given that the first step in maltose catabolism is: α-glucosidase maltose + H2 O −−−−−−→ 2 D-glucose why doesn’t Candida utilis ferment maltose? Kluyver and Custers reasoned ‘that the organism is able to synthesize its numerous different cell compounds from the unsplit disaccharide . . . seems utterly absurd’ [194, p. 132]. Their view was consistent with the findings of Emil Fischer who, at the end of the nineteenth century, had firmly established for yeasts that oligosaccharides are always hydrolysed before they are fermented27 ( [117], and see [28]). Hence, the inability of C. utilis to ferment glucose was not easy to interpret. Indeed, Kluyver and Custers found no lack of α -glucosidase activity in a strain of Kluyveromyces thermotolerans (Torulopsis dattila ), which gave the Kluyver effect with maltose. Working in the late 1930s, they suggested that the effect was caused by anaerobic conditions reversibly inactivating some glycoside hydrolases, such as α -glucosidase [194, p. 159]. On 10 May 1940, the German army invaded Holland, so that Kluyver’s research was severely interrupted for several years [185] and it was not until the 1950s that an alternative explanation became available; namely, inactivation of the mechanism of transport across the plasma membrane. Such an explanation became feasible after Jacques Monod and his colleagues had characterized selective permeation systems, which are responsible for the entry of metabolites into microbial cells (e.g. [300], see [25]). Results of investigating the same problem for maltose utilization by Mucor rouxii in 1969 were ‘interpreted to mean that a functional respiratory chain is required for maltose penetration into the cell’ [119], as had been suggested the previous year for yeasts [16, pp. 566– 567]. Furthermore, in other contexts, there were reports that certain yeasts required oxygen for the transport of sugars into their cells. For example, (a) a non-fermenting yeast, Rhodosporidium toruloides , was found to transport Dglucose actively under aerobic conditions, but not to take up that sugar anaerobically [202], and (b) a 27

In 1922, Richard Willst¨ atter (1872–1942) and Gertrud Oppenheimer (1893–1948) had disputed Fischer’s view [386]. They found that certain yeasts ferment lactose more rapidly than they ferment D-glucose, D-galactose or an equimolar mixture of the two and, hence, concluded that the first metabolic step is not necessarily hydrolytic. Their evidence for ‘direct’ fermentation of oligosaccharides remained a matter of dispute (e.g. [217,218]) until 1949, when Alfred Gottschalk pointed out that the rate of entry of a sugar across the plasma membrane might limit the rate of catabolism of that sugar [143].


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respiratory-deficient mutant of Saccharomyces pastorianus was shown to have a much reduced rate of maltose uptake compared with the wild-type [310].

Observations of Sims and Barnett However, it was not until the late 1970s that Tony Sims 28 and Barnett began investigating the physiology of the Kluyver effect in yeasts [324]. Basing their information on a survey by taxonomists [231], they listed the responses of 100 species which appeared to show the effect for at least one of nine oligosaccharides, commenting: ‘This effect is widespread and possibly at least as common amongst yeasts as the Pasteur effect’. Table 6 gives examples from these authors’ list and illustrates the finding that there was no obvious pattern of occurrence of the Kluyver effect; on the contrary, there was striking individuality among yeasts in their response to each substrate. Sims and Barnett extended the notion of the Kluyver effect to the utilization of D-galactose. The route by which D-galactose is transformed to D-glucose 6-phosphate (see [25]), itself an intermediate of the glycolytic pathway (Figure 7), involves no net oxidation. Hence, there seemed to be no reason for the catabolism of D-galactose to differ from that of D-glucose in its oxygen requirements. These workers studied yeasts which gave this effect with maltose, cellobiose and D-galactose, using a carbon dioxide electrode to measure CO 2 output under both aerobic and anaerobic conditions. A ninefold increase in the rate of CO 2 output occurred only a few seconds after admitting air into an anaerobic suspension of maltose-grown Candida utilis and was immediately linear (Figure 8). The rapidity of the Table 6. Nine yeast species which show the Kluyver effect with more than one sugar: their utilization of certain glycosides and D -galactose (after Sims and Barnett [324]) Species

SUC

MAL

CEL

TRE

LAC

MEL

MLZ

MeG

RAF

Gal

Candida chilensis

K

K

K

+

K

−

K

K

−

K

Candida ergatensis

K

K

+

K

K

−

−

K

−

+

Candida haemulonii

+

+

−

+

−

−

?

−

?

K

Candida utilis

+

K

K

K

−

−

K

K

?

−

Debaryomyces castellii

+

+

+

K

K

+

+

+

+

K

Debaryomyces polymorphus

+

?

?

?

K

K

?

?

+

?

Metschnikowia lunata

K

K

K

+

−

−

K

K

−

K

Pichia heimii

+

K

K

+

−

−

K

K

−

+

Pichia naganishii

K

K

K

+

−

−

K

−

−

−

Pichia strasburgensis

+

K

+

+

−

−

K

+

?

+

K, Kluyver effect, i.e. fermentation negative and aerobic growth positive; + , fermentation and aerobic growth both positive; − , fermentation and aerobic growth both negative; ?, doubt as to how results should be interpreted; SUC, sucrose; MAL, maltose; CEL, cellobiose; TRE, α,α -trehalose; LAC, lactose; MEL, melibiose; MLZ, melezitose; MeG, methyl α -D-glucopyranoside; RAF, raffinose; Gal, D -galactose. Notes: (i) All these yeasts ferment D -glucose to give ethanol and carbon dioxide. (ii) The tests used to provide this information were crude and unquantitative [17]. Those results given as + or − should be repeatable; those in doubt are listed as ?. However, growth rates of, for example, 0.5 or 0.05 generations h −1 might both be registered as + . The results come from [29]. 28

Anthony Peter Sims (1933–1990), English biochemist, worked at the University of East Anglia, Norwich on the regulation of metabolism in Candida utilis, other fungi and green plants [19].


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Figure 7. Routes of galactose and glucose catabolism (Leloir pathway simplified)

Figure 8. Representation of recorder traces for aerobic and anaerobic carbon dioxide output by maltose-grown Candida utilis (NCYC 737). A suspension (0.4 mg dry wt/ml) of C. utilis was carbon-starved aerobically for 2 hours in Difco yeast nitrogen base. 10 ml was transferred to a CO2 electrode chamber and made anaerobic by bubbling with nitrogen. Traces: (i) Negative control : the rate of endogenous CO2 by the yeast was recorded for about 2 min; air was then admitted for about 5 s (↑) and the recording was continued; (ii) endogenous anaerobic CO 2 was recorded; 5 µ mol maltose (MAL ↓) were added at about 2 minutes and air was admitted at about 3 minutes ( O2 ↓), the yeast then again made anaerobic and further recordings were made of anaerobic and aerobic CO2 output. Reproduced from [324]

changes was suggestive of some form of activation and deactivation, rather than the slower processes involving induction or derepression, for which enzymic (or carrier) synthesis is essential (see [101]). Moreover, with C. utilis, which shows the Kluyver effect for the β -glucoside, cellobiose, 29 there was no loss of β -glucosidase activity associated with a change from aerobic to anaerobic conditions [324]. 29

Whereas maltose (4-O-α -D-glucopyranosyl-D-glucopyranose) is an α-linked glucose-glucose disaccharide, cellobiose (4-O-β -Dglucopyranosyl-D-glucopyranose) is the same, but β -linked:


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Since Since inacti inactivit vity y of the hydrolases did not appear appear to explai explain n the Kluyver Kluyver effect, effect, it seemed seemed worth investigating whether the carriers which take sugars into the cells might be deactivated, as had been suggested previously [16,119]. The crude results from tests by taxonomists also indicated that transport might might well well be an import important ant factor factor.. For those those oligos oligosacc acchar haride idess which which are mostly mostly hydrol hydrolyse ysed d in the 30 cytosol (Table 7), 70–100% of the yeasts showed the Kluyver effect. On the other hand, for those usually hydrolysed outside the plasma membrane, the corresponding figure was <35% [324] (Table 8). For sucrose, however, the figure was about 40%: probably this is because sucrose ( β -D-fructofuranosyl α -D-glucopyranoside) is a double glycoside, that is, both a β -fructoside and an α -glucoside. Although most yeasts hydrolyse sucrose with an external invertase, which is a β -fructosidase, many do so with an internal (cytosolic) α-glucosid -glucosidase ase (Figure (Figure 9; for review, review, see [18]). [18]). According Accordingly, ly, Sims and Barnett Barnett measured the rates of entry of sugars into the cells. Kluyveromyces thermotolerans (Torulopsis dattila ) were Result Resultss of experi experimen ments ts with with Kluyve Kluyver’s r’s Kluyveromyces were consistent with the transport of D-[1-3 H]fucose by the D-galactose carrier (Figure 10); and this transport was about four times faster aerobically than anaerobically (Table 9). No such effect was observed with 2-deoxy-D-glucose (2-deoxy- D-arabino-hexose), -hexose), illustrati illustrating ng the important fact that the Kluyver Kluyver effect effect occurs only with certain transport systems in any given yeast.

Figure 9. External hydrolysis of sucrose by invertase and internal hydrolysis by α -glucosidase

Figure 10. Structures of D D -galactose and D -fucose 30

Generally hydrolysed in the cytosol : maltose, cellobiose, lactose, melezitose and methyl α -D-glucopyranoside. Generally hydrolysed outside the plasma membrane: raffinose and melibiose [18].


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Table 7. Location of hydrolysis of oligosaccharides in most yeasts [18]

Table 8. Percentage of species apparently showing the Kluyver effect. Oligosaccharides tested include those usually hydrolysed in the cytosol and others hydrolysed externally to the plasma membrane. Results of a taxonomic survey; the list includes only those yeasts for which all strains are able to (a) grow aerobically aerobically on the specified specified oligosacc oligosaccharid haride e and (b) ferment D -glucose. Data from [29] Oligosaccharides

Percentage of species showing Kluyver effect

Usually hydrolysed in cytosol melezitose lactose methyl α -D-glucopyranoside cellobiose maltose α ,α-trehalose

98 92 87 86 73 54

Usually hydrolysed externally sucrose raffinose melibiose

33 24 14

Entry of maltose into Candida utilis , too, was much slower anaerobically than aerobically. In further work, on the ‘unregulated’ maltose uptake of a mutant 31 of Saccharomyces Saccharomyces cerevisiae , Barnett and Sims found found that that the active active transp transport ort of exogen exogenous ous maltos maltosee ceases ceases on switch switching ing from from aerobi aerobicc to anaero anaerobic bic 31

The mutant was defective in glucose repression and had uncontrolled uptake of maltose [98].


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Ratess of upta uptake ke of D-[1-3 H]fuco H]fucose se by galacgalacTable Table 9. Rate Kluyveromyces thermotoler thermotolerans ans under tose-grown Kluyveromyces under aerobi aerobicc and anaerobic conditions. conditions. Note: the inhibitio inhibition n of uptake uptake by D-galactose is consistent with the entry of D-fucose by the D -galactose carrier. (From [324]) Condit io ion

Inhibitor

Rat e of uptake nmol min−1 (mg dry wt)−1

Aerobic

none

20.7

Anaerobic

none

4.8

Aerobic

D-galactose

1.28

Anaerobic

D-galactose

0.84

ability of a mutant mutant strain strain of Saccharomyces cerevisiae, to concentra concentrate te exogenousl exogenously-supp y-supplied lied maltose. maltose. Figure 11. The ability , Aerobic Aerobic uptake; uptake; ž, anaerobic anaerobic uptake; uptake; - - - - , equilibriu equilibrium m condition conditions, s, when exogenous exogenous and endogenous endogenous maltose maltose concentrations are the same. Reproduced from Barnett and Sims 1982 [32]. The mutant, which was defective in glucose repression, had uncontrolled uptake of maltose [98]

°

conditions so that, consequently, the yeast did not concentrate maltose anaerobically (Figure 11) [32]. They They extend extended ed their their study study of the requirem requirement ent of oxygen oxygen for the active active transp transport ort of sugars sugars into into other other yeasts, using strains of Kluyveromyces marxianus and Debaryomyces polymorphus . Experiment Experimentss with 32 the non-metabolizable analogue of lactose, TMG (methyl 1-thio- β -D-galactopyranoside), showed that these yeasts, too, required an oxygen supply for the active transport of lactose, which Barbara Schulz and Milan Höfer ofer later confirmed for D. polymorphus [321]. Although Barnett and Sims found that active transport ceases under anaerobic conditions, facilitated diffusion, 33 by which which the glycosid glycosides es can also enter enter the cells, cells, seemed seemed to be unaffe unaffecte cted. d. Hence, Hence, they they conclu concluded ded that that the contro controll mechan mechanism ism underl underlyin ying g the Kluyve Kluyverr effec effectt (a) probab probably ly also also acts acts at a later later stage of catabolism, such as in the pathway from pyruvate to ethanol (Figure 12), and (b) is not mediated by the slower processes involving induction or repression [32]. 32

-galactopy topyran ranosid oside) e) was used by Adam Adam Kepes Kepes for studying studying the kinetic kineticss of β -galactosid -galactosidee transport transport into TMG (methyl 1-thio-β -D-galac Escherichia coli in the 1950s [190] (see also [25]). 33 Facilitated diffusion is carrier-mediated movement across a membrane which, unlike active transport, depends on a concentration gradient and not on expenditure of metabolic energy (for review see [91]).


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Figure 12. Anaerobic and aerobic pathways of sugar catabolism in yeasts

Hendrik van Urk and his colleagues found the levels of pyruvate decarboxylase (see Figure 12) in Saccharomyces cerevisiae and Candida utilis to be associated with the rate of catabolic flux in the anaerobic utilization (fermentation) of D-glucose [358]. Observations on six species of yeast by Sims and Barnett were consistent with these findings [325]. Five of these yeasts utilized one or more disaccharides aerobically, but not anaerobically, although all used D-glucose anaerobically, that is, all five showed the Kluyver effect; but the sixth yeast, S. cerevisiae , did not do so. When grown on a glycoside with which it showed the Kluyver effect, each yeast had much less pyruvate decarboxylase activity than when grown on a glycoside with which it did not give the effect (exemplified in Table 10). There was no consistent corresponding lowering of activity of either alcohol dehydrogenase or of the relevant glycosidase. Hence, they concluded, ‘pyruvate decarboxylase may have a role in producing the Kluyver effect’ [325, p. 295] and the chain of events might be as follows: (a) Glycolytic flux may be low as a result of a combination of: (i) The change from active transport to facilitated diffusion, which leads to a low concentration of glycoside in the cytosol and; (ii) The low affinity of the glycosidase for its substrate. 34 (b) The consequent diminution of the rate of glycolysis leads to the rapid deactivation of pyruvate decarboxylase, as described later for Kluyveromyces lactis [37], the enzyme being activated by its substrate, pyruvate [39,174,314,324,335]. (c) While switching to anaerobic conditions activates pyruvate decarboxylase, transport is greatly slowed down by a reduction in the supply of ATP, so pyruvate decarboxylase activation fails because of reduced glycolytic flux.

Experiments of Jack Pronk and his colleagues Although some later work on maltose catabolism by Candida utilis , published by Jack Pronk and his colleagues at Delft in 1994, gave support to the notion that transport limitation is a factor in the Kluyver effect, their findings with pyruvate decarboxylase conflicted with the idea that inactivation of that enzyme was also a factor [378]. They found that pyruvate decarboxylase activities of C. utilis grown on maltose in oxygen-limited culture had a higher flux even than in Saccharomyces cerevisiae under the same conditions. The authors suggest that the Kluyver effect is caused by feedback inhibition of sugar transport by ethanol [379]. 34

Two β -glucosidases of Debaryomyces polymorphus have K m = 22 mM and 40 mM-cellobiose, respectively [361].


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Table 10. Specific activities of three enzymes in two yeasts grown on different sugars as sole source of carbon: expressed as nmol substrate catalysed min−1 (mg protein)−1 . Results of Sims and Barnett [325] Yeast

Carbon source for growth

Pyruvate decarboxylase

Alcohol dehydrogenase

Glycosidasea

Candida viswanathii

D-glucose

0.13

1.04

–

maltose

0.11

0.32

0.33

cellobioseb

0.039

0.23

0.33

D-glucose

1.62

0.57

–

maltose

0.40

0.64

1.01

Saccharomyces cerevisiae

a

Yeasts grown on maltose were tested for α -glucosidase activity. C. viswanathii grown on cellobiose was tested for

β -glucosidase activity. b

C. viswanathii gave the Kluyver effect with cellobiose.

In order to test the hypothesis that yeasts, which show the Kluyver effect for sucrose, hydrolyse it intracellularly [18,324], Pronk and his colleagues investigated sucrose uptake and metabolism by Debaryomyces yamadae [184]. And, indeed, they concluded: The results indicate that the Kluyver effect for sucrose in D. yamadae . . . is effected by rapid down-regulation of the capacity of the sucrose carrier under oxygen-limited conditions [184, p. 1567].

Kluyver effect mutants: fds and gal2 In 1978, working in Norwich with Barnett, Entian attempted to isolate mutants of Kluyveromyces lactis which did not show the Kluyver effect from strains that already did so. Although 40 000 colonies of mutagenized cells grown aerobically on lactose plates were replica-plated onto maltose, cellobiose or α ,α -trehalose (all substrates giving the Kluyver effect with these yeasts), none of the colonies was able to grow anaerobically on these sugars [100]. However, certain mutants failed to grow with glycerol. These fds mutants were totally aerobic and depended entirely on anaerobic fermentation. However, they were not respiratory-deficient and, hence, were similar phenotypically to the glucose derepression mutant snf1 of Saccharomyces cerevisiae (see below). When these mutants were tested against substrates that gave the Kluyver effect, none was utilized aerobically. Poisoning respiration with KCN immediately prevented uptake of these substrates and led to an instant decrease in the concentration of D-glucose 6-phosphate. Adding glucose to these poisoned cells promptly restored fermentation, which showed that glycolysis was still functioning. From these observations and the genetical data, Entian and Barnett concluded: All these results were consistent with the requirement of an energy supply for the transport of maltose, alpha,alphatrehalose or cellobiose, that involved the cytochrome system. [100, p. 325].

In the context of the Kluyver effect, Hiroshi Fukuhara has recently drawn attention to the failure of gal2 mutants of Saccharomyces cerevisiae to use D-galactose anaerobically, although they will grow on it aerobically [123]. (The GAL2 gene encodes the main galactose carrier [2,66,86,87].) Furthermore, introducing a wild-type GAL2 gene into yeast with a gal2 mutant restores the ability to use galactose anaerobically. Results of some genetic experiments with Kluyveromyces lactis also give credence to the theory that loss of the supply of metabolic energy, necessary for transport, has a r oˆ le in producing the Kluyver effect. Paola Goffrini and her colleagues in Parma have been studying the curious case of the Kluyver effect with Copyright © 2005 John Wiley & Sons, Ltd.

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Figure 13. Raffinose

the trisaccharide, raffinose (O-α -D-galactopyranosyl-(1 → 6)- α -D-glucopyranosyl β -D-fructofuranoside, Figure 13). This case is curious, because raffinose is usually hydrolysed outside the plasma membrane by invertase (cf. Figure 9) to produce melibiose and D-fructose. Now K. lactis does not utilize melibiose [29] and the fructose might be expected to be transported into the cells by a hexose carrier, as described for Saccharomyces cerevisiae [65,201]. Hence, given that failure of transport across the membrane is critical for producing the Kluyver effect, raffinose utilization would not be expected to be subject to the effect. However, Goffrini and her colleagues report overcoming the effect in this yeast by introducing sugar carrier genes from S. cerevisiae and they conclude: These results strongly suggest that the sugar uptake step is the major bottleneck in the fermentative assimilation of certain sugars in K. lactis and probably in many other yeasts [139, p. 427].

It is uncertain whether the tight coupling of concentrative monosaccharide transport to aerobic metabolism described for Rhodosporidium toruloides [202], mentioned above, can be compared to mechanisms underlying the Kluyver effect. In any case, a study of both phenomena could well assist the progress towards an understanding of the transport of sugars into yeasts. Furthermore, Pronk and his colleagues have suggested a potential industrial use for the Kluyver effect: Because the use of yeast strains exhibiting a Kluyver effect obviates the need for controlled substrate-feeding strategies to avoid oxygen limitation, such strains should be excellently suited for the production of biomass and growth-related products from low-cost disaccharide-containing feedstocks [51, p. 621].

The Crabtree effect (repression of respiration) Despite glucose repression in yeasts often being called the ‘Crabtree effect’, there are major differences between these two phenomena, and so some explanation is given here of this effect and its history. In the 1920s, following up Warburg’s findings that certain tumour tissues have a higher rate of glycolysis than normal cells [368], Herbert Crabtree35 studied the respiration of tumour cells and found that 35

Herbert Grace Crabtree (1892–1966), English biochemist, was with the Imperial Cancer Research Fund in London for 43 years [6].


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adding glucose decreased the respiration rate [71,175]. Unlike glucose repression in yeasts, the Crabtree effect in tumour cells is commonly explained in terms of a decrease in ADP within the mitochondria [55,292] because ADP is imported into the mitochondria by an exchange with cytoplasmic ATP. If efficient glucose fermentation produces a high concentration of ATP in the cytoplasm, importing of ADP into the mitochondria is prevented, and the consequent depletion of ADP leads to a lower rate of respiration. This, however, does not explain why 2-deoxy- D-glucose (2DG) produces a Crabtree effect [396]. In 1958, Kenneth Ibsen and his colleagues [176] showed that the level of ATP decreases almost immediately after adding 2DG, and the Crabtree effect could be measured within 20 seconds after adding glucose. From these observations, and also because 2DG gives a Crabtree effect too, these authors concluded that the level of cytoplasmic ATP is overcome by a disproportionate reaction in the mitochondria of 2ADP → ATP + ADP, ADP being exported into the cytoplasm. This export decreases the concentration of ADP within the mitochondria. In 1961, Benno Hess and Britton Chance carefully studied the kinetics of the Crabtree effect in tumour cells [163], distinguishing between a short-term Crabtree effect, occurring within 2 minutes of adding glucose, and a long-term Crabtree effect, which occurred after 20–30 minutes. The short-term effect was explained by an excess of ATP within the mitochondria and the long-term effect by reduced import of ADP into the mitochondria. Both result in depleted mitochondrial ADP.

Glucose repression in yeasts The eccentric behaviour of Saccharomyces cerevisiae , when supplied with D-glucose, has already been mentioned in this series [31]: even in air, most of the pyruvate formed by glycolysis is channelled to ethanol, rather than into the tricarboxylic acid cycle (Figure 14) and, accordingly, the yeast’s respiratory activity is decreased. When in 1966 De Deken described the catabolism of glucose by a number of yeast species (Table 4), he named this decrease in respiration, produced by glucose, the ‘Crabtree effect’ — after Crabtree’s findings [78]. However, the physiological reasons for the lower rate of respiration after adding glucose are completely different in yeast and tumour cells. Whereas, as described above, the respiratory decrease in tumour cells depends solely on metabolic changes (ADP depletion), the corresponding respiratory decrease in yeast cells is caused by the repression of the structural genes responsible for synthesizing respiratory enzymes [128]. Hence, the term ‘Crabtree effect’ is a misnomer for glucose repression in yeasts [101]. Glucose repression was first reported for Escherichia coli by Helen Epps and Ernest Gale, who termed it ‘glucose effect’ [111]. Later, Boris Magasanik used the term ‘catabolite repression’ instead [240]: in 1961, he wrote: . . . considerations led us . . . to formulate the concept that catabolites which are formed rapidly from glucose accumulate in the cell and repress the formation of enzymes . . . It is this interpretation of the ‘glucose effect’ which suggests ‘catabolite repression’ as an appropriate name for this phenomenon [240, p. 251].

In 1998, Juana Maria Gancedo explained: When [glucose or fructose] is present, the enzymes required for the utilization of alternative carbon sources are synthesized at low rates or not at all. This phenomenon is known as carbon catabolite repression, or simply catabolite repression, and since no ‘catabolite’ derived from glucose and involved in the repression has been yet identified, the term ‘glucose repression’ has been proposed . . . I still use the term ‘catabolite repression’ as well as glucose repression, to stress that other sugars, such as galactose or maltose, are able to affect the synthesis of enzymes repressed by glucose [129, p. 334].

In 1942, Epps and Gale had described their ‘glucose effect’ as follows: ‘the presence of glucose in the medium during the growth of Escherichia coli suppresses the formation of certain enzymes’ [111]. Copyright © 2005 John Wiley & Sons, Ltd.

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(A)

Figure 14. Diagram of aspects of metabolism of D -glucose and ethanol by Saccharomyces cerevisiae in (A) derepressed and (B) glucose-repressed cells (after Ronne [304])

Solomon Spiegelman and John Reiner in 1947, and Wilbur Swanson 36 and Charles Clifton 37 in 1948, reported a similar finding for Saccharomyces cerevisiae (or S. pastorianus ) [331,342]. In their excellent paper, Spiegelman and Reiner carefully examined the galactose metabolizing pathway, which they referred to as ‘galactozymase’ (see [25]). They observed that a yeast pre-grown with galactose, and thereafter transferred to a glucose-containing medium, lost its galactozymase activity; but this loss was prevented by adding azide 38 (Figure 15). Two years later, azide was shown to inhibit phosphorylation [232]. Studies of glucose repression, described below, have shown that the presence of glucose in the growth medium stops the transcription of glucose-repressible genes. As a consequence, after adding glucose: (a) the total amount of certain enzymes remains constant; (b) however, the specific activity (enzyme activity per mg protein) decreases, because the number of cells that do not transcribe increases [96]. In 1948, Swanson and Clifton gave an account of the effects of glucose repression (although they did not use this expression) in Saccharomyces cerevisiae . When the yeast grew aerobically in batch 36

Wilbur H. Swanson (1903–?) worked with Charles Clifton at the Department of Bacteriology and Experimental Pathology, School of Medicine, Stanford University, California in the 1940s, moving to San Jose State College in 1948. 37 Charles Egolf Clifton (1904–1976), American microbial biochemist, worked at the Department of Bacteriology and Experimental Pathology, School of Medicine, Stanford University from 1929, becoming professor of bacteriology (information kindly supplied by Patricia A. French of Lane Medical Library, Archives and Special Collection Department, Stanford University, School of Medicine). 38 Sodium azide (NaN3 ) prevents the coupling of ADP phosphorylation to aerobic respiration [232]; in 1949, Eugene Kennedy and Albert Lehninger found that isolated mitochondria catalyse oxidative phosphorylation, which is coupled to the oxidation of intermediates of the tricarboxylic acid cycle [189].


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(B)

Figure 14. Continued

Figure 15. Loss of ‘galactozymase’ activity by Saccharomyces sp. on adding D-glucose. The loss was prevented by adding NaN3 . Arrows (↓) indicate when NaN3 was added. ž loss of activity in control ž, activity after adding NaN 3 ; suspension, without NaN3 . Reproduced from Spiegelman and Reiner (1947) [331]; The Journal of General Physiology , 1947, vol. 31 , p. 183, by copyright permission of The Rockefeller University Press


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Figure 16. The tricarboxylic acid and glyoxylate cycles. Reproduced from [197], courtesy of H. L. Kornberg and by permission of Elsevier

culture on 56 m M D-glucose, alcoholic fermentation predominated until the glucose disappeared from the medium [342]. Sixteen years later, Walter Bartley and his colleagues published three key papers (in 1964 and 1965) which described a major step towards understanding glucose repression in Saccharomyces cerevisiae . Sugars in the medium for growing this yeast aerobically caused ‘an anaerobic type of metabolism as measured by ethanol production’, D-glucose being much more effective in this respect than was D-galactose. This glucose repression affected enzymes of the tricarboxylic acid cycle, both glucose and galactose repressing the key enzymes of the glyoxylate cycle 39 (Figure 16) almost completely. Furthermore, glutamate dehydrogenase was more than 50 times more active in S. cerevisiae when it was grown on pyruvate than on D -glucose [287]. In addition, Bartley and his colleagues found no mitochondrial structures in yeast grown aerobically on glucose but, with the removal of glucose, mitochondria reappeared as the yeast’s ability to respire acetate returned [288,289]. And in 1971, Alberto Sols and his colleagues wrote: There is considerable uncertainty as to whether the impairment of respiration caused by glucose is: (i) a case of the ‘catabolite repression’ that affects the synthesis of many catabolic enzymes (Polakis et al., 1965 [289]; DeDeken, 1966 [78]; C. P. M. G o¨ rts, 1967 [141]); (ii) related to the disassembly of normal mitochondrial structures; or (iii) involves a combination of factors. The mechanism(s) of catabolite repression in general is far from clear, and is currently under study in several laboratories [330, p. 301].

Many kinds of enzyme in yeasts have been found to be subject to glucose repression. These include respiratory enzymes [110,337], glyoxylate cycle enzymes [287,389], gluconeogenic enzymes [126,130,148], disaccharide hydrolysing enzymes [85,120,212,341,359,360] and many others (Table 11). 39

Glyoxylate cycle (a modification of the tricarboxylic acid cycle) by which two molecules of acetate form one molecule of C4 dicarboxylic acid, occurs not only in yeasts, but also in bacteria (e.g. [200]), filamentous fungi (e.g. [68,196]) and green plants (e.g. [198]).

Acetate Citrate Oxaloacetate iso Citrate

Malate

Acetate Glyoxylate

Succinate

The glyoxylate cycle was first described by Kornberg, Krebs and Madsen in 1957 [199,205]. In 1960, Barnett and Kornberg published evidence of its occurrence in the yeasts, Kluyveromyces lactis, Saccharomyces cerevisiae and Zygosaccharomyces bailii [27]. However, Schizosaccharomyces pombe is said to lack two key enzymes of the cycle [112], which may explain its reported inability to utilize acetate as sole carbon source for growth [207, p. 345].


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By the mid-1980s it was clear that the underlying regulation of glucose repression in Escherichia coli differed from that in yeasts. Research on E. coli had shown D-glucose to lower the levels of cAMP40 , which nucleotide is necessary for the transcription of genes sensitive to carbon catabolite repression [177,353]. However, evidence was accumulating that this was not true of yeasts [241]. Adding exogenous cAMP to strains of Saccharomyces cerevisiae that were permeable to it did not prevent the repression of galactokinase [246] and levels of cAMP were at least twice as high in repressed S. cerevisiae, Schizosaccharomyces pombe or Kluyveromyces marxianus as in the non-repressed yeasts [112]. Today, it is clear that the molecular mechanism of glucose repression in E. coli differs completely from that in S. cerevisiae. In E. coli , binding of cAMP to the cAMP receptor protein (CRP or CAP 41 ) is necessary for the transcription of glucose repressible enzymes [93,323,400]. By contrast, there is no CRP homologous protein in S. cerevisiae and, unlike E. coli , there is a transient increase in cAMP concentration in S. cerevisiae within 2 minutes of adding glucose [248,291,356]. Indeed, there is no evidence that the level of cAMP in yeasts is associated with the glucose repression of synthesis of enzymes, such as invertase [261].

Genetic analysis of glucose repression and identification of the genes involved This historical review takes into account the following steps in the analysis of gene function: (a) identifying the gene loci involved, by means of isolating mutants; (b) isolating the respective genes, in most cases by plasmid complementation of the respective mutants; (c) sequencing the genes; 42 and (d) characterizing the biochemical function of the proteins encoded by each gene.

Nomenclature of genes and their synonyms There are many synonyms for the genes involved in regulating glucose repression and it is difficult to decide which name should be used for each gene. Genetic convention is to prefer the name used in the first description of a mutant. However, many mutants which proved to be synonymous were isolated independently. Often their allelism was demonstrated much later than their original description, in many cases only after their respective wild-types had been isolated and sequenced. Accordingly, the chosen name of each gene could be that used when it was first sequenced. Mark Johnston has suggested that, like the nomenclature of mitochondrial proteins, the glucoserepression community should decide on new names, each of which would refer to the gene’s function. Although this idea is attractive, this historical survey is no place to generate further confusion with yet more names. Accordingly, in order to help follow the complexities of the molecular-genetic control of glucose repression, the large number of pleiotropic genes involved and their synonyms are summarized in Table 12 (see below). 43 In the early 1970s, several repression mutants were described. One of these, flk1, was highly pleiotropic: for this mutant, invertase, α-glucosidase and flocculent growth were each non-repressible [310]. Earlier, Oliver Lampen and his colleagues had described a mutant with a similar phenotype, but had only characterized it biochemically [137,261]. Mutant flk1 was found later to be allelic to tup1, whose function is described below in Table 13. 40

cAMP, cyclic AMP, adenosine 3 , 5 -cyclic monophosphate, is formed from ATP in a reaction catalysed by adenylate cyclase and has regulatory functions in many kinds of organism. cAMP was first reported in a yeast in 1966 [56]. 41 The cAMP receptor protein is also named CAP (for catabolite gene activator protein). 42

Understanding the molecular basis of glucose repression became possible in the 1970s with the development of methods of gene isolation and sequencing. The first yeast gene was probably cloned in 1976 [338] and yeast transformations were reported in 1978 [35,166]. Accordingly, in the 1980s, many genes corresponding to glucose-repression mutants were isolated and their sequences determined. 43 For ease of reading, gene synonyms are given in brackets where original findings are reported and, thereafter, preference is given to the gene name which has been first sequenced.


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Table 11. Repression by D -glucose of some enzymes in yeasts Enzyme repressed

Yeast

Repression (%)

Conditions

ACONITATE HYDRATASE

S. cerevisiae

51

Comparing growth in 50 m M D-glucose with that in 44 m M pyruvate [287]

P. anomala S. cerevisiae

39 91

Grown in 167 mM D-glucose; then incubated for 4 h in (a) 56 m M D-glucose or (b) 61 mM NaAc; (a) compared with (b) [88]

87

Comparing growth in 167 mM D-glucose with that in 0.43 M ethanol [384]

K. marxianus

>99

Comparing growth in 111 mM D-glucose with that in 11 m M D-glucose [75]

S. cerevisiae

>99

Comparing growth in different concentrations of D -glucose, from 250 m M to 3 M [85]

91


C. salmanticensis D. carsonii D. hansenii P. anomala P. membranifaciens Rh. glutinis Rh. minuta Rh. mucilaginosa Rhs. toruloides Sp. pararoseus S. cerevisiae

>98


β -GALACTOSIDASE

K. marxianus

>99


β -GLUCOSIDASE

K. marxianus × K. dobzhanskii

∼90

Comparing growth in 1 m M D-glucose with that in 0.1 m M D-glucose [239]

GLUTAMATE DEHYDROGENASE

S. cerevisiae

50


ISOCITRATE DEHYDROGENASE NAD+

S. cerevisiae

59


30


67


(aconitase) ALCOHOL DEHYDROGENASE

β -FRUCTOFURANOSIDASE

(invertase or inulinase)

S. pastorianus FRUCTOSE BISPHOSPHATASE

(

69 62 82 90 86 52 89 76 95 >98

)

ISOCITRATE DEHYDROGENASE (NADP+ )

S. cerevisiae

In 1975 Michael Ciriacy 44 (Figure 17) devised an electrophoretic system by which he could distinguish three isoenzymes of alcohol dehydrogenase: alcohol dehydrogenase I (Adh1p 45 ), alcohol dehydrogenase II (Adh2p) and mitochondrial alcohol dehydrogenase (Adh3p). He showed that Adh1p is mainly present 44

Michael Ciriacy (1947–1996), German geneticist, studied the regulation of alcohol dehydrogenase isoenzymes, showed the first Ty1 retrotransposon integration to be responsible for constitutive adh2 expression, and characterized glucose carriers of Saccharomyces cerevisiae genetically. He was in Fritz Zimmermann’s laboratory at Darmstadt from 1977 to 1981, when he became a professor at the Institute of Microbiology at the University of Düsseldorf [160]. 45 Abbreviations used for proteins, for which each gene is responsible, are written as the abbreviation of gene’s name, printed in roman type, with the first letter a capital, e.g. Adh1. This may also be written Adh1p: the ‘p’ is added (for protein) to prevent misunderstanding. This convention differs from that used for the genes; e.g. the wild-type structural gene of alcohol dehydrogenase I is written ADH1 (in italic capitals) and a mutant is adh1 (italic lower case).


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Table 11. Continued ISOCITRATE LYASE

MALATE DEHYDROGENASE

MALATE SYNTHASE

OLIGO-1,6-GLUCOSIDASE

P. anomala Rh. glutinis S. cerevisiae

94 99 99


99

Comparing growth in 83 mM D-glucose with that in 61 m M NaAc [389]

P. anomala

3


S. cerevisiae

44 87

P. anomala Rh. glutinis S. cerevisiae

S. pastorianus

94 99 95


73

Comparing growth in 50 mM D-glucose with 44 m M pyruvate [287]

99


87

Comparing growth in 111 m M D-glucose with that in 17 m M D-glucose [310]

67 55 50 5 10 >65 9–75 63 79


(isomaltase) PHOSPHOENOLPYRUVATE CARBOXYKINASE

C. pelliculosa Cr. humicola P. anomala Rh. glutinis Rh. mucilaginosa S. cerevisiae S. pastorianus Z. fermentati Z. rouxii


C, Candida; Cr, Cryptococcus; D, Debaryomyces; K, Kluyveromyces; P, Pichia; Rh, Rhodotorula; Rhs, Rhodosporidium; S, Saccharomyces; Sp, Sporidiobolus; Z, Zygosaccharomyces

during growth with glucose and is the major enzyme involved in ethanol production, whereas Adh2p is subject to glucose repression [60,61]. He also described a mutation of the ADH2 promoter46 which made alcohol dehydrogenase II 47 insensitive to glucose repression [62]. Molecular analysis showed that this insensitivity was caused by a promoter insertion of the yeast transposon Ty1 [384]. The first pleiotropic 48 mutants of glucose repression, cat1 and cat2, were isolated by Fritz Zimmermann in 1977 [398] by screening for mutants that could grow on glucose, but not on ethanol, as the carbon source. The cat1 mutants failed to derepress various enzymes: α -glucosidase, invertase, and also gluconeogenic and respiratory enzymes; hence, these mutants did not grow with ethanol, maltose or sucrose as a sole source of carbon. That same year, Ciriacy, who later joined Zimmermann’s laboratory, established a system for selecting mutants in which there was no glucose repression. Ciriacy used haploid mutants lacking a constitutive alcohol dehydrogenase 49 and, from these haploid mutant strains, he selected new mutants which could not 46

A promoter is a DNA region upstream to the coding sequence of a gene, which binds RNA polymerase. Alcohol dehydrogenase II (Adh2p), encoded by the gene ADH2, catalyses the first step of gluconeogenesis from ethanol. Adh2p is cytoplasmic, necessary for alcohol degradation and is repressed by glucose several hundred-fold [236]. 48 A pleiotropic mutation has more than one phenotypic effect. 49 Alcohol dehydrogenase I (Adh1p), unlike Adh2p, is the enzyme responsible for the formation of ethanol in ‘alcoholic fermentation’. 47


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Figure 17. Michael Ciriacy

Figure 18. Connexion between the glyoxylate and tricarboxylic acid cycles (modified from a diagram by Kornberg and Madsen, published in 1957 [199])

grow on glycerol or ethanol [63]. These new mutants included ccr1, in which there was no derepression of (a) enzymes of gluconeogenesis, (b) isocitrate lyase (of the glyoxylate cycle; see Figures 16 and 18) or (c) fructose bisphosphatase 50 (Figure 19), that is, the strains carrying ccr1 could not synthesize these enzymes, whether or not glucose was present. Ciriacy’s ccr1 mutant was allelic to Zimmermann’s cat1 50

Fructose bisphosphatase ( D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) is often written ‘fructose-1,6-bisphosphatase’ in order to distinguish it clearly from fructose-2,6-bisphosphate 2-phosphatase, EC 3.1.3.46, (also written ‘fructose-2,6-bisphosphatase’). Fructose bisphosphatase catalyses D -fructose 1,6-bisphosphate + H2 O → D -fructose 6-bisphosphate + ortho-phosphate. Fructose bisphosphatase was first prepared from kidney and liver in 1943 by George Gomori [140] and its specificity for fructose 1,6-bisphosphate was reported in 1955 [250].


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Figure 19. The regulation of glycolysis by activators and deactivators

(Ciriacy, personal communication) as well as to the snf1 mutant (Schüller and Entian, unpublished), isolated by Marian Carlson [48] (see Table 12). The cat1 mutation affects all the glucose-repressible enzymes and, as described below, later biochemical analysis has shown that cat1 encodes the most central element in the regulatory circuit of glucose repression, a protein kinase, named Snf1-4-kinase (see below).

Zimmermann’s selection system for mutants defective in glucose repression A further advance in the genetic analysis of glucose repression was Zimmermann’s development in 1977 of a powerful system for selecting mutants which resisted glucose repression [399] (described in an earlier article in this series [25]). Working with Saccharomyces cerevisiae growing exponentially on glucose as carbon source, he plated this yeast on medium containing low concentrations (0.6–1.8 m M) of 2-deoxy- D-glucose (2DG) plus raffinose. The selection of mutants depended on certain properties of 2DG: 1. S. cerevisiae (and other yeasts), although using D-glucose, does not use 2DG for growth; 2DG is toxic, as it is phosphorylated and incorporated into the cell wall, which becomes severely damaged [162,180,181]. 2. S. cerevisiae hydrolyses the raffinose by means of invertase [387,388], to give melibiose and D -fructose, of which only fructose is utilized. 3. S. cerevisiae takes up D-glucose, D-fructose and 2DG by the same carriers [65,201]. Copyright © 2005 John Wiley & Sons, Ltd.

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Table 12. Genetic and biochemical characterization of genes involved in glucose repression description Gene

Mutant isolation

Mutant phenotypes

Gene sequence

∗

Asterisk indicates first

ˆ of wild-type protein Physiological r ole

Subunits of the central Snf/Cat complex

CAT1

Zimmermann and colleagues 1977∗ [398]

CCR1 Ciriacy 1977 [63] SNF1 Carlson and colleagues 1981 [48] CAT3

Entian and Zimmermann 1982∗ [108]

SNF4 Neigeborn and Carlson 1987 [271] SIP1

No growth on non-fermentable Celenza and carbon sources; no derepression of Carlson α -glucosidase, invertase, 1986∗ [52] gluconeogenic or glyoxylate cycle enzymes Zimmermann and colleagues 1977 [398]

Catalytic subunit of a Ser/Thr-specific protein kinase complex, phosphorylates activators and repressors involved in glucose repression. Celenza and Carlson 1986∗ and 1989 [52,53]

No growth on non-fermentable Schüller and carbon sources; no derepression of Entian 1988∗ α -glucosidase, invertase, [319] gluconeogenic or glyoxylate cycle enzymes Entian and Zimmermann 1982 [108] Celenza and colleagues 1989 [54]

γ -Subunit of the Ser/Thr-specific

Yeast two-hybrid interaction with Snf1-kinase Yang and colleagues 1992 [394]

β -Subunits (scaffold proteins) of the

Snf1/Cat1 kinase Celenza and colleagues 1989 ∗ [54] Partial nuclear localization Schüller and Entian 1988 ∗ [319]

Yang and colleagues 1992∗ [394]

SIP2


GAL83


Ser/Thr-specific Snf1/Cat1 kinase Yang and colleagues 1994 ∗ [395]

Gene repressors under control of Snf/Cat complex

MIG1

Nehlin and Ronne 1990∗ [270] CAT4 Schüller and Entian 1991 [320] SSN1 Vallier and Carlson 1994 [355]

Multicopy inhibitor of GAL gene induction. Nehlin and Ronne 1990 [270]. No repression of invertase or α -glucosidase; increased hexokinase P11 activity Schüller and Entian 1991 [320]

Nehlin and Ronne 1990∗ [270]

C2 H2 Zinc-finger protein binds as repressor to most glucose-repressible genes Nehlin and Ronne 1990 ∗ [270] Released from the nucleus upon Snf/Catcatalysed phosphorylation DeVit and colleagues 1997∗ [82]

Gene repressors not under control of Snf/Cat complex

MIG2

Lutfiyya and Johnston 1996 ∗ [235]

Overexpression of MIG2 represses Lutfiyya and Johnston 1996 ∗ SUC2 [235]

C2 H2 Zinc-finger protein binds to Mig1p binding site, not a target of Snf/Cat kinase Lutfiyya and colleagues 1998 ∗ [234]

Zimmermann found that glucose-grown wild-type cells take up the toxic 2DG but, because the invertase is glucose-repressed, do not hydrolyse the raffinose. Spontaneous mutants occur in all populations; those Copyright © 2005 John Wiley & Sons, Ltd.

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Table 12. Continued Gene activators under control of Snf/Cat complex

CAT8

DIL1

SIP4

Hedges and colleagues 1995∗ [158] Rahner and colleagues 1996 [295] Lesage and colleagues 1996∗ [221]

No derepression of gluconeogenic or glyoxylate cycle enzymes. Fails to grow with ethanol as carbon source. No derepression of isocitrate lyase

Hedges and colleagues 1995∗ [158] Rahner and colleagues 1996 [295]

Interacts with Snf1 in the yeast two-hybrid system

Lesage and colleagues 1996∗ [221]

Binuclear zinc cluster ( Zn2 Cys6 ) gene activator binds to CSRE elements Hedges and colleagues 1995 ∗ [158], Rahner and colleagues 1999 [294] Activation through Snf/Cat phosphorylation Randez-Gil and colleagues 1997 ∗ [296] Binuclear zinc cluster ( Zn2 Cys6 ) gene activator binds to CSRE elements. Activated through Snf/Cat phosphorylation. Vincent and Carlson 1998∗ [362]

Subunits of the Glc7 phosphatase

CID1

GLC7

HEX2

REG1

Neigeborn and Carlson 1987 [271]

Constitutive invertase synthesis

Peng and colleagues 1990 [284] Entian and Zimmermann 1980∗ [107]

Does not accumulate glycogen

Matsumoto and colleagues 1983 [246]

Glc7 type 1 protein phosphatase Tu and Carlson 1994 ∗ [351]. Dephosphorylates Snf1 at Thr-210 McCartney and Schmidt 2001∗ [249] Feng and colleagues 1991∗ [114] Niederacher and Entian 1991∗ [273]

No repression of invertase or α -glucosidase; partial derepression of respiratory enzymes; increased hexokinase PII activity, inhibited by maltose No repression of galactokinase

Subunit of Glc7 Tu and Carlson 1995 ∗ [352]

Other proteins involved in the signalling of glucose repression

HEX1

HXK2

CAT80

GRR1

Entian and colleagues 1977∗ [109]

Lobo and Maitra 1977∗ [229]

Entian and colleagues 1977∗ [109]

¨ Fr ohlich and colleagues 1985∗ [121]

No repression of invertase or α -glucosidase; partial derepression of respiratory enzymes; decreased hexokinase activity Reduced glucos e phosphorylation

No repression of invertase or α -glucosidase; abnormal cell shape

Bailey and Woodword 1984 [14]

Flick and Johnston 1991 ∗ [118]

Hexokinase PII gene. Possibly important in signalling glucose repression Entian 1980∗ [97], Entian and Mecke 1982 [106]. Phosphorylated through Snf/Cat complex Randez-Gil and colleagues 1998 ∗ [297,298] Possibly indirect effect on glucose-repression; part of a ubiquitin-conjugating enzyme complex which regulates Rgt1p, a regulator of certain hexose transporters ( HXT -genes) Li and Johnston 1997 ∗ [222]

which have non-repressible invertase can hydrolyse the raffinose to give melibiose and D-fructose. So such mutants become supplied with an excess of exogenous fructose molecules, which competitively prevent the uptake of 2DG (Entian, unpublished). Hence, those cells that grew in a medium containing Copyright © 2005 John Wiley & Sons, Ltd.

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2DG + raffinose were derepressed mutants which had high invertase activity, even when glucose was the carbon source. Table 12 lists genes and their mutants which are involved in glucose repression and derepression and summarizes the phenotypic effects of the mutations. The mutants, hex1 [399], hex2 and cat80 [109,399], were isolated using Zimmermann’s selection system. These mutants all had pleiotropic effects on glucose repression, and their functional analysis (described below) showed all three genes, HEX1, HEX2 and CAT80, to be central components of the regulatory circuit of glucose repression. Each of these three mutants affected the glucose repression of invertase, α -glucosidase and respiratory enzymes, but not the repression of gluconeogenic enzymes. Later, in the 1980s, various other methods led to the isolation of mutants which were allelic to those mutants that Entian and Zimmermann had obtained in 1977 and 1980 [107,399]. Mutant glr1 (allelic to hex1 ) was selected by using glucosamine instead of 2-deoxy- D-glucose [259]; reg1 (allelic to hex2 ) was isolated using a selection system for non-repressible galactokinase [246]; and grr1 (allelic to cat80 ) [14] was selected on medium containing 0.1 M D-galactose and 0.6 m M 2DG.

ˆ in glucose repression Entian’s analysis of hexokinases and their r ole When analysing the way these non-repressible mutants functioned, Entian appreciated that the pattern of derepression strongly resembled that of wild-type cells during growth on galactose, which had been carefully examined by Polakis and Bartley in 1965 [287]. Hexokinase is the only glycolytic enzyme that is by-passed during growth on galactose (see Figure 12 of article 7 in this series [25]) and the regulatory rôle of the genes was underlined, when enzymic analysis revealed that hexokinase activity was much decreased in hex1 and significantly increased in hex2 mutants [107,109,206,278]. The original cat80 mutant showed normal hexokinase activity but, with a different genetic background, the grr1 mutation (which was allelic to cat80 ) gave a three-fold higher hexokinase activity [14]. Also in the 1970s, Pabitra Maitra 51 and Zita Lobo52 carried out a careful genetic analysis of glucose phosphorylation in Saccharomyces cerevisiae . They identified the loci of the structural genes for the hexokinase isoenzymes PI and PII and their respective mutants, hxk1 and hxk2, as well as the glucokinase mutant, glk1 [243]. Their system differed from that of Zimmermann: they selected mutants with an increasing resistance to higher concentrations of 2-deoxy- D-glucose than he had used, the cells being resistant to 2DG because of their failure to phosphorylate this sugar [228–230,242]. Saccharomyces cerevisiae , it should be explained, possesses three hexose-ATP-kinases, namely (a) two hexokinases (both EC 2.7.1.1), PI and PII 53 [69], which phosphorylate both D-glucose and D-fructose and (b) the D-glucose-specific glucokinase (EC 2.7.1.12) [242,244]. The relative rates of activity of PI and PII differ: the rate of PI with D-fructose is about three times that with D-glucose, whereas PII gives nearly the same rates with both substrates [215,393]. By the 1980s, the biochemistry of yeast hexokinases had been studied extensively (see [99] for review) and, using the mutants of Lobo and Maitra, Entian was able to show that (a) the hex1 mutant corresponded to the hexokinase PII structural gene hxk2 [97,106] (Table 12) and (b) HXK2 was markedly overexpressed in hex2 mutants. These findings indicated a regulatory as well as a catalytic function for hexokinase PII, which was found to be important for triggering glucose repression [103,106]. After identifying hexokinase PII as a key enzyme in glucose repression, Entian wrote: As shown previously . . . the lowered hexokinase activity [in hex1-mutants] was not associated with reduced metabolite levels. This agrees with the similar catalytic activities of mutant and wild-type hexokinases at low substrate concentrations . . . We hypothesize that, in addition to its catalytic activity, hexokinase PII also has a regulatory component. This of course requires that the enzyme changes considerably, depending on the availability of hexoses or their catabolic derivatives [97 p. 637]. 51

Pabitra K. Maitra (b. 1932), Indian biochemist and geneticist, worked at the Tata Institute of Fundamental Research in Bombay (Mumbai). Together with Zita Lobo, he made major contributions to the genetics and biochemistry of yeast carbohydrate metabolism. 52 Zita Lobo (?–2000), Indian biochemist, was married to P. K. Maitra, with whom she published most of her 23 papers on carbohydrate metabolism and genetics of yeasts [303]. 53 In the 1960s, Saccharomyces cerevisiae was found to have two distinct hexokinase isoenzymes, PI and PII [135,215,322].


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It is still not known how hexokinase PII acts on the regulatory system. The original hypothesis of an enzyme with a dual function, both catalytic and regulatory [97,106], has been neither proved nor disproved. Entian and his colleagues have isolated hexokinase PII mutants with defective glucose repression which, nonetheless, maintain their catalytic activity [103,104] but these mutants have not been characterized at a molecular level. Furthermore, overexpression of the structural gene for yeast glucokinase, GLK1 (which is only 40% homologous with the structural genes for yeast hexokinases I and II), did not restore glucose repression. Hence the hexokinases, themselves, do have a specific regulatory role. On the other hand, David Botstein and his colleagues obtained mutants which gave evidence of a marked association between hexokinase catalytic activity and glucose repression [238] and similar results were obtained after domain swapping experiments between hexokinase isoenzymes PI and PII [305]. So, thus far, the question remains unresolved.

Carlson’s analysis of sucrose-non-fermenting (snf ) mutants The pleiotropic hex2 mutants were sensitive to maltose, so that adding maltose to a culture of such mutant strains inhibited their growth, glycolysis and protein synthesis [98]. This effect of maltose on these mutants was the consequence of its uncontrolled uptake and hydrolysis, which produced a high intracellular concentration of glucose [98,105]. Entian exploited this maltose toxicity, as it provided a convenient system for the selective isolation of mutants which are specifically required for maltose utilization, as well as others involved in general carbon catabolite repression. The cat3 mutant, reported in 1982 [108], was almost identical phenotypically to the cat1 mutant described in 1977 [398]. Later, CAT1 and CAT3 were shown to encode major components of a multimeric protein kinase complex and the epistasis of hex2 (= reg 1) is explained, as this gene encodes a subunit of the counteracting phosphatase Glc7p [351,352]. Also during the 1980s, Carlson and Botstein began another systematic investigation into glucose repression in Saccharomyces cerevisiae . They and their colleagues isolated a large number of mutants which failed to utilize sucrose and were named snf mutants (for sucrose non fermenter) [48]. Two of these mutants, snf1 and snf4, proved to be of major importance towards furthering the understanding of the molecular mechanism which underlies glucose repression. The snf1 mutants were shown to be allelic to cat1 and the snf4 mutants to cat3 (Table 12 gives the synonyms of some of these alleles). Various findings were important for further augmenting the understanding of the molecular control of glucose repression: Carlson’s showing that SNF1 encodes a protein kinase [52,53] was particularly important. Sequencing established that the CAT3 [319] and SNF4 genes [54] are identical. The Cat3p protein was located in the nucleus [54,319]. Co-immunoprecipitation experiments showed that SNF1 and SNF4 form a common protein complex (Snf1p–Snf4p)54 [54]. 5. Epistasis was observed of cat1 (= snf1 ) and cat3 (= snf4 ) mutants over the hex2 (= reg1 ) mutation [108]. 6. It was found that reg1 (= hex2 ), a subunit of the GCL7 , encodes protein phosphatase 1 55 [351,352]. 1. 2. 3. 4.

Further major advances became practicable with the establishment of the yeast two-hybrid genetic system for studying interactions between proteins (Figure 20), described in 1989 by Stan Fields and 54

The physical interaction of SNF1 and SNF4 was used to establish the yeast two-hybrid system [116], which is even today an effective method for obtaining first indications of physical protein–protein interactions and is used in functional genome analyses for many species. 55 GCL7 is an essential gene. Originally, a mutant with non glucose-repressible invertase was isolated and called cid1 (for constitutive invertase derepression) [271]. Later analysis of this mutant showed it to be viable (T152K ) within the GLC7 gene [351].


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Figure 20. Diagram of the two-hybrid system of Fields and Song (based on their Figure 1) [116]. The GAL4 protein is a transcriptional activator, which expresses genes encoding enzymes of the galactose pathway. (1) The GAL4 protein consists of two separable domains, which do not function when separated: (a) Gal4-BD is a domain which binds specific DNA sequences (UASG ); and (b) Gal4-AD is a domain which activates gene transcription. (2) Fields and Song separated these two domains as two genes and (3) fused Gal4-BD to protein X and Gal4-AD to protein Y. (4) If X and Y interact to form a dimer, the two domains are brought together and transcription is activated

Ok-kyu Song [116]. 56 This system has become widely used for selecting proteins that interact with a known protein. By means of yeast two-hybrid screenings, Carlson’s group used Snf1p to identify three other proteins, Sip1, Sip2 and Gal83, which interact with the Snf/Cat kinase [53,394,395], and domain interaction analyses, using the same system, indicated that these three (Sip1p, Sip2p and Gal83p) act as alternative scaffold proteins. 57 Under conditions of glucose repression, these scaffold proteins build bridges; Gal83p, for example, is the bridge between Snf1p and Snf4p and, under derepressing (low glucose) conditions, this bridge may support the direct interaction between the now phosphorylated Snf1p and its positively acting regulatory protein Snf4p (Figure 21) [179]. 58 The scaffold subunits are also responsible for the intracellular location of the Snf/Cat complex [363]. The major scaffold proteins for glucose repression seem to be Gal83p, because it directs the Snf/Cat kinase complex with its nuclear localizing sequence (NLS) to the nucleus [363], and Sip2, because it is N -myristoylated [12] 59 and therefore retains the Snf/Cat complex in the cytoplasm due to its fixation at the plasma membrane [223]. 56

Fields and Song made use of the characteristics of the transcriptional activator protein GAL4p of Saccharomyces cerevisiae [116]. This activator has functional domains for DNA binding (Gal4p-BD) and for gene activation (Gal4p-AD), which Fields and Song separated as two genes. Gal4p-BD is fused to one protein, X, and Gal4p-AD to protein Y. If X and Y interact to form a dimer, this dimerization brings the Gal4p-AD and Gal4p-BD together. As a result, transcription of genes regulated by GAL4p DNA-binding sites is activated and the activation can be detected. This system has made it practicable to identify and clone genes, the products of which interact with a known protein of special interest [59]. The known protein is fused to Gal4p-BD and expressed in a gal4 deletion strain. Libraries of these fusions are screened for clones which activate a GAL4-regulated promoter. 57 The S. cerevisiae Snf/Cat kinase is a homologue of the highly conserved AMP-activated serine/threonine kinases, which are found in plants, Drosophila, Caenorhabditis elegans, mammals and fungi (for review, see [154]). The Snf/Cat kinase contains a catalytic α-subunit encoded by SNF1, a regulatory γ subunit encoded by CAT3 (SNF4 ), and the β -subunits, which act as scaffold proteins , having structural rˆ oles as temporary structural frameworks but no catalytic properties. 58 This interpretation of the genetic results of yeast two-hybrid analysis was recently confirmed biochemically, using ‘tandem-affinitypurification’ and MALDI– TOF– MS (mass spectrometry) to analyse the protein composition of protein complexes [134]. The N-terminal glycine residue of Sip2 is modified by myristoylation [12]. 59 Protein N -myristoylation promotes weak and reversible protein–membrane interactions [283].


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Figure 21. Diagram of protein interactions involved in regulating glucose repression in Saccharomyces cerevisiae (after Carlson [47]). Events in low glucose concentration: (1) scaffold proteins bring Snf1p kinase and Snf4p protein together; (2) Snf4p protein activates Snf1p kinase; (3) Snf1 kinase permits transcription of glucose-repressed genes

Repressors and activators under regulatory control of the Snf/Cat kinase The major repressor, Mig1p, that binds to glucose-repressible proteins, was isolated and characterized in the early 1990s by Hans Ronne and his colleagues [269,270]. They found that the mig1 mutants reversed glucose repression for certain genes, including SUC2 (encoding invertase) as well as the GAL1 and GAL4 genes (encoding galactokinase and the GAL gene specific activator Gal4p). At about the same time, the cat4 and the ssn1 mutants were isolated as suppressors of the expression of (a) α -glucosidase (‘maltase’) in cat1 and cat3 mutant strains [320] and (b) invertase [355]. Finding these mutants to be allelic to mig1 (H.-J. Schüller, personal communication) provided the first indication that Mig1p and the Snf/Cat kinase are interrelated. Although binding sites for the Mig1p repressor were detected in nearly all glucose-repressible genes, those genes concerned with gluconeogenesis were unaffected. This difference in response to mig1 mutants was explained after Entian and his colleagues had identified the Cat8p gene activator (zinc cluster 60 ) protein [158,294], which was also found as DIL1 when Hans-Joachim Schüller and his colleagues screened for mutants that failed to derepress the gluconeogenic enzyme, isocitrate lyase, encoded by ICL1 [295]. Cat8p is necessary specifically for the transcription of gluconeogenic genes and, hence, is essential for growing yeast on non-fermentable carbon sources. Recent use of microarray analyses has confirmed this highly specific function of Cat8p with about 30 target genes, of which 12 are strongly regulated by Cat8p. These include all the structural genes of the enzymes of gluconeogenesis [156,290]. The CAT8 gene, itself, is under the regulatory control of Mig1p and, hence, its expression is repressed on glucose. However, expression of CAT8 is not sufficient for the transcription of gluconeogenic genes, because the Cat8 protein needs post-translational activation via Snf/Cat kinasemediated phosphorylation [296]. The existence of this dual control system for the expression of gluconeogenic genes explains why all attempts, even with highly stringent selection systems, have failed [252]. 60

Zinc cluster proteins : some regulators of transcription contain a zinc cluster (or zinc finger) which enables them to bind to specific DNA sequences. For example, X-ray crystallography has shown that Gal4p binds to certain DNA sequences, CGG N11 CCG, with each zinc cluster recognizing a CGG triplet. Many DNA-binding proteins have a zinc cluster, which is a polypeptide chain bound to a zinc atom.


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Figure 22. Regulation of Class I glucose-repressible genes, such as the structural genes for SUC2 and CAT8. If glucose is present in the medium, the Mig1 repressor binds to the respective URS sites (upstream repressing sequences) of Class I genes and prevents their transcription. Under these conditions, Snf/Cat kinase is inactive. If glucose is exhausted in the medium, the Snf/Cat kinase is activated and phosphorylates the Mig1 repressor (probably with the nuclear Snf/Cat–Gal83 complex kinase). Consequently, the phosphorylated Mig1 repressor is exported to the cytoplasm. It is not known whether Mig1 phosphorylation is a prerequisite for the dissociation of Mig1 from the URS of the respective structural genes

Another important step towards understanding the physiology of glucose repression has been the finding by Johnston and his colleagues that the Mig1p repressor is exported from the nucleus, within minutes, when cells are derepressed by depriving them of exogenous glucose [82]. A series of observations, summarized below in this paragraph, have provided the basis for understanding the rôles of the Snf/Cat kinase and Mig1p in glucose repression (see Figures 22 and 23). As mentioned above, mig1 and its allelic isolates, cat4 [320] and ssn1 [355], partly suppressed the snf1 mutant, indicating that the Snf1p kinase deactivates Mig1p. Snf/Cat kinase has been shown to phosphorylate Mig1p at least in vitro [276,327,348]. Furthermore, Johnston and his fellow authors have given convincing evidence that Mig1p phosphorylation brings about nuclear exclusion of Mig1p by means of binding the nuclear exportin Msn5p [81], which had been previously identified genetically as a multi-copy suppressor of Snf1p [113]. In order to exert repression, Mig1p requires the additional interaction with the repressors Cyc8p (= Ssn6p) and Tup1p; hence cyc8 (= ssn6 ) and tup1 mutants fail to repress respiratory enzymes and/or invertase [49,307,349]. Mutations within both these genes have pleiotropic effects and, having first been identified by virtue of such different effects, these mutants have been given several synonymous designations, which are listed in Table 13. Cyc8p and Tup1p, physically associated within a large nuclear protein complex [383], interfere with chromatin structure [92] and both of them act as mediator proteins for other regulatory proteins, which explains their pleiotropic effects (for review, see [328]). Copyright © 2005 John Wiley & Sons, Ltd.

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Figure 23. Regulation of Class II glucose-repressible genes, such as the structural genes for gluconeogenesis and the glyoxylate cycle. If glucose is exhausted in the medium, the Snf/Cat kinase is activated and has a dual function. First, Snf/Cat kinase phosphorylates the Mig1 repressor (probably catalysed by the nuclear Snf/Cat–Gal83 complex). Consequently, the phosphorylated Mig1 repressor is exported to the cytoplasm. Second, Snf/Cat kinase phosphorylates the Cat8 gene activator (probably catalysed by the plasma membrane-bound Snf/Cat–Sip2 complex). NB: the CAT8 structural gene is also a Class I glucose-repressible gene under Mig1 control. In order to function, the CAT8 gene must be released from glucose repression; its post-translational activation is via the Snf/Cat kinase

The current model of glucose repression: single and double61 control systems To sum up, the mechanism of glucose repression is now quite well understood and may be described simply as follows. The Snf/Cat kinase is the central element for glucose repression and regulates the activity of the respective gene repressors and activators. Under conditions of glucose limitation, the Snf/Cat kinase complex is activated when it is phosphorylated. The catalytically active Snf/Cat kinase complex then has a dual function: 1. It phosphorylates the Mig1p repressor, so inhibiting its action, and enabling it to be exported from the nucleus. 2. By means of phosphorylation, it activates specific gene activators such as Cat8p. 61

Double control system : two systems, and if one fails the other one will take over.


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Table 13. Pleiotropic effects of mutants of the genes CYC8 and TUP1 Mutant

cyc8

tup1

Allelic designations

Phenotype

cyc8

Over-expression of iso-2-cytochrome c [307] Producing non-repressible invertase [349]

ssn6

Extragenic suppressor of a snf1 ( cat1) mutation [49]

tup1

Ability to take up thymidine [381] Constitutive invertase synthesis on glucose [349]

flk1

a

Flocculent growth; the first genetically characterized mutant with non-repressible invertase, α -glucosidase and galactose utilization [310,332]

umr7

Resistant to UV-induced CAN1 mutations [219]

amm1

Stabilizing ARSa -defective plasmids [345]

cyc9

Over-expression of iso-2-cytochrome c [307]

aar1

α -Specific mating type defect [153,262]

aer2

Increased expression of CYC1, CYC7 (iso-2-cytochrome c ) and GAL1, [397]

sfl2

Flocculation phenotype [122]

ARS, autonomous replicating sequence necessary for chromosome and plasmid replication.

Transcriptional Repressor

Glucose

Glucose-repressible Structural Genes

Snf/C at kinase complex

Transcriptional Activator

Figure 24. Simplified diagram of the regulation of glucose repression. The Snf/Cat kinase is inactive if glucose is available in the medium. Hence, the Snf/Cat complex has a dual function in regulating glucose repression. If activated, when no glucose is available, Snf/Cat kinase (a) inactivates the transcriptional repressor and (b) activates the transcriptional activator; so that finally the transcriptional repressor dissociates from the glucose-repressible structural genes and the activator binds to the structural gene

Figure 24 gives a simplified picture of these events. Three kinases, Pak1p, Tos3p and Elm1, can activate the Snf/Cat kinase [170,265,340] from which the Pak1 kinase seems to derive its major physiological function, as Pak1-dependent phosphorylation of Snf/Catp is important for the nuclear localization of the Snf/Cat–Gal83 complex [157]. It is still not known how the kinases that activate the Snf/Cat kinase complex are, themselves, activated when glucose is depleted, or how hexokinase PII interferes with the system. Copyright © 2005 John Wiley & Sons, Ltd.

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Three findings in the late 1990s have provided further evidence for a regulatory role of hexokinase PII in glucose repression. First, hexokinase PII is present in the nucleus as well as in the cytoplasm [297]; second, there is an Snf1p-dependent phosphorylation of hexokinase PII [296]; and third, hexokinase has been identified as a target for Hex2p-dependent phosphorylation [3]. These three findings are evidence of the interrelation of hexokinase PII and the mechanism of glucose repression. However, it is still unclear whether the effects observed with hexokinase PII are responsible for, or just the consequence of, glucose-dependent regulation.

Classification of glucose-repressible genes according to their regulation Three classes of glucose-repressible genes have been recognized: (I) genes whose expression is under a single glucose control mechanism; (II) genes whose expression is under a single glucose control mechanism, but which also require induction; and (III) genes which are under additional glucose control mechanisms. The expression of Class I genes mainly depends on the release of the Mig1p repressor from the respective promoters and can be described as follows. After its Snf/Cat1-dependent phosphorylation, Mig1p is then exported from the nucleus and derepresses several genes, which include the invertase structural (SUC ) genes and gene activators such as CAT8 (Figure 22). Expression of Class II genes requires the release of Mig1p from the promoter and the additional binding of an inducible gene activator. This class (II) of genes includes those concerned with catabolizing maltose ( MAL genes) and galactose ( GAL genes). Class III genes are strictly controlled by glucose. Their expression depends on (a) the release of Mig1p from the promoter and (b) the binding of specific gene activators. Class III genes are mainly those belonging to crucial metabolic pathways for the utilization of non-fermentable carbon sources, such as those catabolized via gluconeogenesis and the glyoxylate cycle. The specific gene activator is CAT8, a class I gene. However, transcription of CAT8 is, of itself, insufficient for transcribing the genes concerned with gluconeogenesis. Furthermore, the Cat8 protein is activated by phosphorylation, which is catalysed by Snf/Cat kinase. In other words, the dual control of class III genes prevents failure of glucose repression of the gluconeogenic enzymes. And such failure would, of course, be disastrous for the cell. After its activation, Cat8p also binds to its functional homologue Sip4p [221], which then enforces derepression of gluconeogenic genes (Figure 23).

Enzyme inactivation and the regulation of gluconeogenesis A yeast growing on a non-fermentable source of carbon, such as acetate, ethanol, glycerol or lactate, requires high activities of enzymes of both the gluconeogenic pathway (Figure 19) and the glyoxylate cycle (Figures 16 and 18). Adding D-glucose to such a yeast leads to the onset of glycolysis and to high activity of fructose bisphosphatase. Without regulation of the enzyme activity of the two reciprocal pathways (glycolysis and gluconeogenesis), an energy-wasting ‘futile cycle’ would ensue, between phosphofructokinase and fructose bisphosphatase (see Figure 19). Accordingly, certain enzymes are strictly regulated by several biochemical and genetic systems, which are dependent on the nature of the available carbon source. These enzymes include both the two key enzymes of gluconeogenesis (phosphoenolpyruvate carboxykinase and fructose bisphosphatase, encoded by genes PCK1 and FBP1 ) and the enzymes of the glyoxylate cycle (isocitrate lyase, malate synthase and cytoplasmic malate dehydrogenase, encoded by genes ICL1, MLS1 and MDH2, respectively). In addition to allosteric inhibition of fructose bisphosphatase by both AMP [126] and D-fructose 2,6-bisphosphate 62 [132], the transcription of all genes which encode the gluconeogenic and glyoxylate cycle enzymes are 62

D-fructose 2,6-bisphosphate was demonstrated in Saccharomyces cerevisiae in 1981 [216].


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subject to glucose repression (see above). Furthermore, in 1965 Helmut Holzer 63 (Figure 25) and his fellow-workers had found that malate dehydrogenase activity rapidly disappears on adding D-glucose to the medium [390], and this crucial finding drew attention to another important mechanism that regulates the amount of enzyme in the cell, that is, the specific proteolysis of enzymes 64 (see also above, Table 2). This specific proteolysis of enzymes, which occurs when glucose is added to yeast cells, has been studied extensively for many years and is called glucose (or catabolite) inactivation (Figure 26) [168]. As long ago as 1947, Spiegelman and Reiner had found that adding D-glucose to cells of Saccharomyces species grown on D -galactose not only repressed but also rapidly inactivated the synthesis of ‘galactozymase’ (the galactose catabolizing pathway) [331]. This inactivation occurred within 4 hours of adding glucose. A similar result was reported in the 1950s by J. J. Robertson and Harlyn Halvorson, who found that the ability of maltose-grown S. cerevisiae to ferment maltose to ethanol and carbon dioxide was stopped about 3 hours after adding D-glucose, although enzymic activity and glucose fermentation were not destroyed [302]. Confirming their conclusion that it was the maltose uptake system which was inactivated, Coen G o¨ rts65 found that adding maltose did not prevent this inactivation [142]; hence, it was the presence of glucose and not the absence of maltose which caused the inactivation. Recovery of maltose uptake after about 1 hour in glucose-free, maltose-containing medium was inhibited by adding cycloheximide, which prevented protein

Figure 25. Helmut Holzer. Photo courtesy of Karl Decker 63

Helmut Holzer (1921–1997), German biochemist, was a pioneer in the study of enzyme regulation. When he was aged 18, World War II began, and he was obliged to undertake labour ( Arbeitdienst ) constructing fortifications along the Rhine. He was in the German army in France and then was wounded in Russia. Holzer joined Feodor Lynen’s group at Munich in 1945 to work on the metabolism of butanol in yeast cells, moving to Hamburg in 1953 and thence to become professor of biochemistry at Freiburg-im-Breisgau in 1956 [77]. 64 Several of these mechanisms, particularly induction, were considered in article number 7 of this series. 65 Coen P. M. G örts (b. 1935), Dutch microbial biochemist, worked in the Botanical Laboratory, State University, Utrecht, The Netherlands, from 1961 to 1972, when he moved to the education department. (C. P. M. G¨ orts, personal communication).


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Figure 26. Catabolite inactivation, as illustrated by Helmut Holzer in 1976 [168]. Reprinted from Trends in Biochemical Sciences 1 : 178–181, copyright 1976, with permission from Elsevier

synthesis [354]. Spiegelman and Reiner had observed earlier that the galactose pathway (‘galactozymase’) was inactivated only when cells were suspended in buffer; not when they were growing [331]. Later, Holzer and his colleagues showed that this inactivation was due to a reduced substrate affinity of the galactose carrier [245].

Holzer’s analyses of glucose inactivation (catabolite inactivation) A different kind of inactivation was found for malate dehydrogenase [390]; its cytoplasmic isoenzyme was rapidly inactivated within 30–60 minutes after adding glucose, even when the cells were growing [90,115]. Similar findings were obtained for various other enzymes, namely, isocitrate lyase [89], fructose bisphosphatase [125], phosphoenolpyruvate carboxykinase [127,148] and 2-isopropylmalate synthase (of the leucine biosynthetic pathway) [45]. Adding cycloheximide 66 did not prevent the inactivation of fructose bisphosphatase [125] or phosphoenolpyruvate carboxykinase [127]; hence, glucose inactivation seemed to be independent of synthesis of the enzymes de novo. However, cycloheximide did prevent inactivation of cytoplasmic malate dehydrogenase [115] but did not prevent this same inactivation in a tryptophan-auxotrophic mutant [90], either in starved cells [268] or in a temperature-sensitive mutant at elevated temperature [267]. These observations provided evidence that protein synthesis de novo is not needed for glucose inactivation of cytoplasmic malate dehydrogenase. Holzer called this phenomenon ‘catabolite inactivation’ but, since it was shown that no glycolytic catabolite occurring after D-glucose 6-phosphate is necessary, the term ‘glucose inactivation’ seemed more appropriate [102]. Cycloheximide, however, prevented the recovery of all these enzymes, which suggested that they were irreversibly degraded proteolytically, and Dieter Mecke and his colleagues provided direct evidence for this interpretation: they showed immunologically that, on adding glucose, the amount of cytoplasmic malate dehydrogenase decreased in proportion to the enzymic activity [149,266]. This finding was also confirmed for fructose bisphosphatase [124] and phosphoenolpyruvate carboxykinase [264]. Further evidence of a proteolytic degradation came from the observation that phenylmethanesulphonyl fluoride 66

Cycloheximide is often used as an inhibitor of protein synthesis, but it is reported to inhibit glycolysis markedly [145].


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(PMSF), an inhibitor of serine-proteases, 67 prevented the inactivation of α-isopropylmalate synthase in permeabilized cells [45] and also prevented the inactivation of isocitrate lyase and fructose bisphosphatase in normal (non-permeabilized) cells [146]. What happens in glucose inactivation seemed even odder: Holzer explained that, when studying inactivation of fructose bisphosphatase, the results of some of their control experiments were inconsistent with there being an irreversible degradation of the enzyme. He and his colleagues showed that adding glucose to cells in their stationary phase (after growth) caused a very rapid loss of 50% of enzyme activity within 1 or 2 minutes. Astonishingly, this rapid inactivation appeared reversible for more than 15 minutes (after adding glucose) as, during that time, the activity could be recovered, even in the presence of cycloheximide (Figure 27) [220]; moreover, experiments with antibodies showed that the enzyme was still cross-reacting during that period [346]. This rapid reversible inactivation is brought about by an enzyme conversion, in which fructose bisphosphatase is phosphorylated [263,247] at serine residue 11 [301]. In vitro, the phosphorylation could be catalysed with a cAMP-dependent protein kinase and the phosphorylation was faster when fructose-2,6-bisphosphatase was present [133]. In vivo, a marked cAMP increase occurred within 2 minutes of adding glucose [347], while the concentration of D-fructose 1,6bisphosphate increased greatly [216]. Proton ionophores also triggered the phosphorylation of fructose bisphosphatase, showing that the membrane potential is important for the increase in cAMP [248]. The finding that fructose bisphosphatase was phosphorylated before its irreversible degradation was, at first, thought to indicate that this conversion triggered the proteolysis of this enzyme. However, Entian and Matthias Rose showed that a mutated form of fructose bisphosphatase, where Ser [11] was replaced by Ala [11], no longer underwent rapidly reversible inactivation, but was still susceptible to irreversible glucose inactivation [306]. Commenting on this work, the Gancedos wrote: It can be concluded that phosphorylation of FbPase is not required for the irreversible inactivation and could be an independent mechanism of regulation, as phosphorylation increases the sensitivity of FbPase towards the inhibitors fructose-2,6-bisphosphate and AMP [131, p. 367].

Isocitrate lyase was also shown to decrease in activity, reversibly, after phosphorylation [233].

Figure 27. Reactivation of fructose bisphosphatase activity after inactivation by glucose. At zero time, 3 and 30 minutes after adding glucose, cells were washed and resuspended in 0.1 M potassium phosphate buffer (pH 6.0) with or without cycloheximide. Reproduced from [220] by permission of Elsevier 67

Proteases (or proteinases), a term originally used in 1928 by Wolfgang Grassmann (1898–1978) and Hanns Dyckerhoff (1904–1965) [144], are orthodoxly called ‘peptidases’ or ‘peptide hydrolases’; this term applies to any enzymes that hydrolyse peptide bonds [274].


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Genetic analysis of glucose inactivation Although the rapid reversible inactivation of some gluconeogenic enzymes after phosphorylation is physiologically significant, the findings described above do not explain how glucose inactivation occurs, or how it is catalysed. Using mutants with blocked glycolysis [64], rapid reversible and irreversible glucose inactivation were found generally in glucose-6-phosphate isomerase ( pgi ), triose-phosphate isomerase (tpi ) and phosphoglycerate kinase ( pgk ) mutants [102]. This clearly showed that no metabolites occurring after glucose 6-phosphate in the glycolytic pathway are needed to trigger both types of inactivation. Unexpectedly, mutants of phosphoglycerate mutase ( pgm ) and pyruvate kinase ( pgk ) showed a rapid reversible inactivation only, but there was no irreversible inactivation. This was explained in terms of interference of the triosephosphates with the proteolytic machinery. Under anaerobic conditions, rapid reversible inactivation was normal in all glycolytic block mutants, but irreversible inactivation did not occur without the expenditure of respiratory energy. Irreversible inactivation was prevented both in a pyruvate kinase ( pyk ) mutant under all conditions and in glycolytic block mutants anaerobically; this rapid inactivation remained reversible, even for as long as 2 hours after adding glucose [102]. These findings provide striking evidence for a proteolytic degradation pathway and research has been focused on identifying the proteases which were responsible for this degradation. Proteases in yeast were first identified and characterized biochemically; and protease A 68 was one of the first enzymes to be described in yeast [136,80,385]. In the early 1980s all known yeast proteases were located inside the vacuole; an exception being aminopeptidase B, which was within the vacuolar membrane. Of these, protease A is of major importance, because it is necessary for the maturation 69 of protease B, carboxypeptidase Y and aminopeptidase I. 70 Mutation within the structural gene of each of these three proteases only eliminated the activity of the respective protease itself. Accordingly, such mutants were used to test whether or not these proteases are involved in glucose inactivation of gluconeogenic enzymes; however, such tests gave conflicting results. Hui-Ling Chiang and Randy Schekman reported the importing of fructose bisphosphatase into so-called ‘Vid vesicles’ (diameter 30–40 nm) and the subsequent vacuolar inactivation of this enzyme [57]. They also isolated vid mutants, defective in vacuolar inactivation; the inactivation depended on protease A and other proteins, such as Vid22p, Cpr1p and Vid24p (Figure 28) [43,44,58]. By contrast to the above findings, Dieter Wolf and his colleagues have reported that, in mutants of protease A and protease B, catabolite inactivation of fructose bisphosphatase is independent of vacuolar proteolysis [251,344,392]; and they have provided clear evidence that inactivation of fructose bisphosphatase requires polyubiquitinylation 71 at the cytosolic 26 S proteasome 72 [315–317]. Although isolating mutants defective in glucose inactivation has proved exceedingly difficult because there were no appropriate selection systems, Entian and his colleagues partly solved this problem in the 1990s. They found that the N-terminal fragment of fructose bisphosphatase, which is composed of 291 N-terminal amino acid residues, is necessary for glucose inactivation and that a fusion of this fragment with E. coli β -galactosidase makes the β -galactosidase susceptible to glucose inactivation. By means of 68

Protease A was purified and described in 1980 [256]; originally designated EC 3.4.23.6, later 3.4.23.25, and named saccharopepsin, yeast endopeptidase A or Saccharomyces aspartic proteinase [377,391]. 69 These proteases are primarily translated as larger proteins from which the active proteases are matured after proteolytic cleavage. 70 This became clear after isolating mutants defective for (a) protease A (pep4 ) [155], (b) protease B ( prb1 ) [161,183], (c) carboxypeptidase Y ( prc1 ) and (d) carboxypeptidase S (cps1 ); for review see [34,164,165]; for activities of several proteases (protease A, protease B, and carboxypeptidase Y), see [182,350]. 71 Ubiquitin is a small protein (8.5 kDa) which attaches to proteins as a preliminary to their destruction in proteasomes. Ubiquitin has been described as ‘the cellular equivalent of the ‘black spot’ of Robert Louis Stevenson’s Treasure Island : the signal for death’ [36, p. 635]. 72 Proteasomes, which occur in the yeast cytoplasm and nucleus, are nanocompartments, where proteolysis is confined. The term was first used in 1988 for particles in HeLa cells [11], and described as very large peptidases with several non-identical subunits and, later, as 20 S cylindrical particles in various eukaryotes [193,343]. In order to move proteins to a proteasome, they are polyubiqutinylated by means of an enzyme cascade, consisting of an ubiquitin-activating enzyme, ubiquitin-conjugating enzymes and ubiquitin– protein ligases which mark these proteins for degradation [34,164,165].


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Figure 28. The concept of fructose bisphosphatase (FBPase) inactivation in the vacuole, by means of Vid vesicles (adapted from [43]). When starved cells of Saccharomyces cerevisiae are given D-glucose, FBPase is taken into Vid vesicles and then degraded in the vacuole. The first step involves at least two cytosolic proteins, Ssa2p and Cpr1p, the level of the latter being regulated by the plasma membrane protein Vid22p. Formation of Vid vesicles is thought to be regulated by the ubiquitin conjunction enzyme Ubc1p. Delivery of FBPase by Vid vesicles to the vacuole depends on Vid24p. Vacuolar proteinase degrades the FBPase

this FBPase–lacZ fusion, these authors established a screening system for isolating mutants which had a defect in glucose inactivation, so that it became practicable to isolate three independent gid -mutants (glucose inactivation deficient, gid1, gid2 and gid3 ).73 All gid -mutants were defective in the glucose inactivation of several enzymes, namely, cytoplasmic malate dehydrogenase, isocitrate lyase and phosphoenolpyruvate decarboxylase, but other functions of the proteasome were unaffected [152]. Of particular interest was finding the N -terminal proline residue to be essential for inactivating fructose bisphosphatase, and a replacement with any other amino acid residue made this enzyme resistant to glucose inactivation. Hence, the N -terminal proline residue is essential for polyubiquitinylation and proteasomal degradation [152]. That cytoplasmic malate dehydrogenase and isocitrate lyase also share a conserved proline residue at the N -terminus provides further evidence for its importance for glucose inactivation. A major advance in the understanding of glucose inactivation was the cloning of the GID3 gene and finding that it was identical with UBC8, which encodes a ubiquitinconjugating enzyme. Like fructose bisphosphatase, Ubc8p is cytoplasmic, and vesicle isolation and proteinase degradation experiments also showed fructose bisphosphatase to be degraded in the cytoplasm under these inactivation conditions [318].

Glucose inactivation: proteasomal versus vacuolar degradation Currently, two distinguished research groups are publishing controversial observations on the molecular mechanism of the enzyme degradation which is triggered by glucose. Either the enzymes are degraded in the vacuole (Chiang and his colleagues) or they are degraded in proteosomes (Wolf and his colleagues). Both groups publish biochemical and genetic observations which support their own respective hypotheses. These apparently conflicting results might conceivably be explained by the existence of more than one pathway for degrading those enzymes which are subject to glucose inactivation. Indeed, there is evidence that the degradation of the galactose carrier (Gal2p) differs from that of fructose bisphosphatase [171]. 73

The gid mutants were analysed further by Michael Thumm, Dieter Wolf and their colleagues; see also [33].


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For galactose, the events may be described as follows. The galactose carrier is first ubiquitinated, then undergoes endocytosis and, finally, is degraded in the vacuole [172,173]. As already mentioned in Spiegelman’s original paper of 1947, inactivation of the galactose carrier occurs only in resting cells, not in growing cells [331]. On the other hand, glucose inactivation of gluconeogenic enzymes occurs in growing cells only. There is strong biochemical and genetic evidence that gluconeogenic enzymes are degraded in the proteasomes of growing cells: in several mutants of the proteosomal pathway, gluconeogenic enzymes were not inactivated. This is so, in particular, in the case of the gid3-mutant of the ubiquitin-conjugating enzyme Ubc8p and has been found more recently with the gid6 -mutant of the de-ubiquitinating enzyme Ubp14p, which prevents the inhibition of proteasomal function [299]. Systematic screening of the mutant deletion collection, EUROSCARF, 74 led unexpectedly to finding several mutants which affect the vacuolar degradation of fructose bisphosphatase [299]. These mutants include Vid24p, a peripheral membrane protein at vesicles (Vid vesicles). These vesicles are thought to transport fructose bisphosphatase to the vacuole (Figure 28) [58]. The involvement of Vid24p in the proteasomal and the vacuolar pathways does not seem to be consistent with the glucose inactivation of deficient phenotype vidp24 mutants. However, this dual function — in the two pathways — is not a general feature of vid mutants, as other vid mutants, such as vid22 and vid27 , which are involved in vacuolar fructose bisphosphatase degradation [43,44], show a typical proteasomal inactivation of this enzyme [299]. Future research will show whether there are, indeed, two alternative pathways for glucoseinduced degradation of gluconeogenic enzymes. Current work suggests that proteasomal degradation is the major pathway when Saccharomyces cerevisiae is growing, whereas the vacuolar degradation pathway may apply when the cells are starved. Although at first an esoteric subject, the specific proteolysis of proteins became a major field of research for understanding cellular regulation. In 1996 Wolfgang Hilt and Dieter Wolf wrote: It is obvious that the few substrates of proteasomes discovered to date represent only the tip of the iceberg and it will be a great challenge to uncover all the cellular processes that proteasomes are involved in, as well as the detailed mechanisms underlying these selective processes [164, p. 101].

This kind of work is likely to have major impacts outside yeast research. An obvious example is that of proteasome inhibitors, which are undergoing clinical trials for the chemotherapy of some cancers [1,138,260], since proteasomes occur in a wide range of organisms, including man [34].

Conclusion The Pasteur, Kluyver and Custers effects are responses by yeasts to changes in the amount or character of the sugars available to them. Enzymic regulation, induction, repression and inactivation bring about these ‘effects’ and make possible other adaptations to alterations in the supplies of nutrients. The early research on microbial adaptations, from 1900 onwards, was physiological and particularly concerned with enzyme induction, which depends on which particular sugar is accessible to the microbe. However, with the development of microbial genetics and molecular biology in the second half of the twentieth century, it became possible to examine the molecular genetics underlying these regulatory phenomena. The first major molecular analysis of a microbial adaptation, carried out during the 1960s to 1980s, was that of the induction and repression of enzymes of the galactose pathway in Saccharomyces cerevisiae . The complex interactions of proteins produced by the various GAL genes, such as activation by Gal4p or repression by Gal80p, were elucidated by the 1990s and this work is discussed in article number 7 of the present series [25]. Towards the end of twentieth century, further complexities of the molecular control of sugar metabolism have been unravelled. Such work has depended on both the development of DNA transformation 74

EUROSCARF, the European S accharomyces cerevisiae Archive for Functional Analysis, contains deletion mutants of all yeast genes (http://www.srd-biotec.de/euroscarf or http://web.uni-frankfurt.de/fb15/mikro/euroscarf/ ).


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systems in yeasts in the late 1970s, first achieved by Jean Beggs [35] and Gerry Fink [166], and also on recombinant DNA technology generally, such as by the creation of yeast vector systems, initially by David Botstein [41] and Kevin Struhl [339]. Many other techniques have contributed to the spectacular advances described above. Immunological methods, such as immunoblotting [167] and immunofluorescence microscopy [57] in the 1990s, have made it practicable to follow the movements of regulatory proteins across various membrane barriers in the cell. One of the main mechanisms by which microbes adapt to changes is by regulating gene expression, and, as understanding what genes do is an essential part of molecular biology, this subject has made many biochemical phenomena in general, and microbial adaptation in particular, more comprehensible. The rôles of certain proteases, kinases and phosphorylations in regulating enzymic activities provide some examples. Because these processes are complex, involve a number of interacting genes and proteins and are in many cases compartmented, the processes are often difficult for non-molecular biologists to follow. Research on the molecular biology of cellular regulation is very active today and will undoubtedly bring to light even greater complexities. Since the entire genome of Saccharomyces cerevisiae has been sequenced (the first eukaryote for which this was done), deletion mutants are available for all approximately 6000 yeast genes [138a]. Consequently, there is enormous progress in understanding the molecular biology of yeast as a eukaryotic model system. New techniques, such as transcriptome analysis [214], the genome-wide yeast two-hybrid analysis [176a] and the use of MALDI MS for proteome analysis of the analysis of TAP-purified protein [134], all generate huge amounts of information which can only be handled by modern bioinformatic methods.

Acknowledgements The authors thank the following most warmly for their help: Joan Brown, Evelyne Dubois, Dylan Edwards, Robert Hauer, Reginald Hems, C. T. Kluyver, Peter K o¨ tter, Matthias Rose and W. A. Scheffers. In addition, they thank L. K. Barnett for all her work towards greatly improving both the writing and illustrations. J. A. B. also thanks the Royal Society for a research grant.

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