Yeast metabolism

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Yeast

Yeast2005; 22: 835894.

Published online in Wiley InterScience (www.interscience.wiley.com).DOI:10.1002/yea.1249

Review

A history of research on yeasts 9: regulation of sugarmetabolism1

James A. Barnett* and Karl-Dieter Entian

School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UKInstitut fur Mikrobiologie, Universitat Frankfurt, Marie-Curie-Strae 9, D-60439 Frankfurt/Main, Germany

*Correspondence to:James A. Barnett, School ofBiological Sciences, University ofEast Anglia, Norwich NR4

7TJ, UK.E-mail: [email protected]

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

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 836The Pasteur effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 836

Pasteurs observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 837Studies by Meyerhof, Warburg and others: 1920s and 1930s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8386-Phosphofructokinase: Engelhardt and Sakov . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 840Saccharomyces cerevisiae and the Pasteur effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 842

The Custers effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 845The Kluyver effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 847

Kluyvers observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 847Observations of Sims and Barnett . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 849Experiments of Jack Pronk and his colleagues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 854Kluyver effect mutants: fds and gal2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 855

The Crabtree effect (repression of respiration) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 856Glucose repression in yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 857Genetic analysis of glucose repression and identification of the genes involved . . . . . . . . . . . . . . . . . . 861

Nomenclature of genes and their synonyms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 861Zimmermanns selection system for mutants defective in glucose repression. . . . . . . . . . . . . . . . . . 865Entians analysis of hexokinases and their role in glucose repression . . . . . . . . . . . . . . . . . . . . . . . . . 868

Carlsons analysis of sucrose-non-fermenting (snf) mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 869Repressors and activators under regulatory control of the Snf/Cat kinase . . . . . . . . . . . . . . . . . . . . . 871

The current model of glucose repression: single and double control systems . . . . . . . . . . . . . . . . . . . . 873Classification of glucose-repressible genes according to their regulation . . . . . . . . . . . . . . . . . . . . . . 875

Enzyme inactivation and the regulation of gluconeogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 875Holzers analyses of glucose inactivation (catabolite inactivation) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 877Genetic analysis of glucose inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 879Glucose inactivation: proteasomal versus vacuolar degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 880

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 881References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 882

1 Previous articles in this series: [2126,28,30,31].

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

Introduction

The present article continues the description of the history of research on induction and repression ofindividual 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 regulatoryprocesses in yeasts. Some account is also given below of the investigation of certain well-known, generalregulatory mechanisms that control sugar metabolism, and which involve enzyme induction, repressionand inactivation.

These mechanisms which regulate sugar metabolism have been called the Pasteur, Kluyver, Custersand Crabtree effects (naming the scientists who first described the respective phenomena), glucose orcatabolite repression, and glucose or catabolite inactivation (Table 1). What has been called the Crabtreeeffect in yeasts should, as will be discussed below, be called glucose repression. Such regulatoryeffects involve enzyme synthesis and enzyme activity (Table 2). In describing the original findings

and the development of later research, an attempt is made to give clear definitions of the phenomenadescribed, as well as an exposition of their physiological roles in Saccharomyces cerevisiae and, asfar as is known, in other yeasts too. An account is also given of the history of research on theinter-regulation of glycolysis and gluconeogenesis.2 The story of studying these processes, like manyaspects of microbiology, began with the work of Louis Pasteur3 who, in 1861, described how thegrowth of yeast per gram of sugar consumed was much greater under aerobic than anaerobic conditions[281].

The Pasteur effect

Many kinds of cell utilize exogenously-supplied sugar faster under anaerobic than under aerobicconditions. This is the Pasteur effect. However, the term has been used variously and the effect hasbeen reported as occurring in many different organisms and tissues. There has been a great deal ofconfused writing on the subject, as indicated in the three quotations from the 1930s which follow thisparagraph, and the large numbers of publications on this topic have been reviewed extensively (e.g.[46,83,84,203,226,293,329,336]).

The intimate relations between the two processes [oxidation and fermentation] has occupied many biochemists sincePasteur discovered their quantitative interdependence, now known as the Pasteur Reaction. Pasteur found that therewas 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 hasbeen one of the most attractive puzzles of biochemistry ever since (Albert von Szent-Gyorgyi 1937 [365, p. 166]).

By far the great majority [of experts on the Pasteur effect] . . . belong to a class which, vaguely aware of thePasteur effect . . . rather accidentally obtain some sort of Pasteur effect, often with some special organism and setof conditions, and announce boldly, not infrequently inNature (or in the good old days, Naturwissenschaften), thathere is the explanation of the Pasteur effect. It is this human, indeed lovable, but mathematically-impossible-that-they-could-all-be-right class that we must be wary of (Dean Burk4 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-Gyorgyi [24], O. Warburg [23].4 Dean Burk (19041988), American biochemist, worked at University College London, the Kaiser Wilhelm Institute in Berlin and

Harvard and Cornell Universities. He became chief chemist at the National Cancer Institute, Bethesda [8].

Copyright 2005 John Wiley & Sons, Ltd. Yeast2005;22: 835 894.


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History of research on yeasts 9 837

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]

CUSTERS EFFECT Brettanomycesand

Dekkeraspp. 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 NADH: NAD+ ratio inhibits

glycolysis

Custers 1940 [73], Wik en and

colleagues 1961 [382], Scheffers

1961, 1966 [312,313]

KLUYVER EFFECT Ability to use

oligosaccharide orgalactose 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 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

REPRESSION

(glucose effect,

carbon catabolite

repression)

Repression of respiration Repression of structural genes of

respiratory enzymes

Spiegelman and Reiner 1947 [331],

Magasanik 1961 [240], Bartley and

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]

GLUCOSE

INACTIVATION

(catabolite

inactivation)

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], Hammerle

and colleagues 1998 [152], Schule

and colleagues 2000 [318]

As considerable confusion exists in the current literature as to the real nature of the Pasteur effect, it is necessary

to explain Pasteurs original conceptions and to describe his experimental results on the effect of oxygen oncarbohydrate catabolism (Kendal Dixon5 1937 [84, p. 432]).

Pasteurs observations

The first relevant publication was that of Pasteur himself, who in 1861 found the growth yield of brewersyeast, 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 manykinds of cell which are capable of both aerobic and anaerobic metabolism. Pasteur put 100 cm3 of sugar

5 Kendal Cartwright Dixon(19111990), 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)

GALand 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 act iv ity of

enzyme after removing

repressing substrate

Genes encoding glucose-

repressible enzymes

Release from repression due

to de-binding of gene

repressors

solution with a little protein into a 250 cm3 flask and boiled the solution to remove the oxygen. Aftercooling, he introduced a very small amount of beer yeast and placed the drawn-out neck of the flaskunder mercury (see [22]). The yeast grew only a little and the sugar was fermented: 60 to 80 parts ofsugar 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 thesame 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, only4 to 10 parts of sugar are transformed. The yeast nevertheless retains its capacity to cause fermentation. Indeedfermentation 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 Pasteurs observations, in the 1920s Otto Meyerhof and Otto Warburg examined differencesbetween 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 comparedthe respiration rates of certain rat cancer cells with those of normal rat cells [372,376]. He found thecancer cells to have (a) the same rate of oxygen consumption as the normal cells but (b) a much higher

6 Si lexperience est faite au contact de lair et sur une grande surface . . .Pour la meme quantite de sucre disparu, il se fait beaucoup plus

de levure. Lair en contact cede de loxygene qui est absorbe par la levure. Celle-ci se developpe energiquement, mais son caractere de

ferment tend a disparatre dans ces conditions. On trouve en effet que pour 1 partie de lev ure formee, il ny aura que 4 a 10 parties de

sucre transforme. Le role de ferment de cette levure subsiste neanmoins et se montre meme fort exalte si lon vient a la faire agir sur le

sucre en dehors de linfluence du gaz oxygene libre [282, p. 80].



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rate of lactate formation, even in the presence of oxygen. In addition, ethyl isocyanide, which inhibitedheavy-metal catalysis of certain oxidations, abolished the slowing down of glycolysis by oxygen. Fromsuch observations, he concluded:

Respiration and fermentation are thus connected by a chemical reaction, which I call the Pasteur reaction after itsdiscoverer.7

Working with both yeast and muscle, it was Meyerhof who was the first to examine Pasteurs observationsof differences between the aerobic and anaerobic breakdown of carbohydrate. Meyerhof found thatglycogen 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 someyeasts is greater in the absence than in the presence of air [258]. He measured oxygen uptake and carbondioxide output, using Warburg manometers,8 and estimated the quotients QO2 and QCO2,

9 both withwashed yeast at 25 C in phosphate solution (0.1 M KH2PO4)and with a high concentration of D-glucose(0.28 M).

A brewers bottom yeast had about the same rate of oxygen uptake, whether in buffer alone or whensupplied with glucose, and the high rate of carbon dioxide production was similar (QCO2 >200) in airor under nitrogen (Table VA of [258]). This finding of Meyerhofs has since been reported many timesfor strains ofSaccharomyces 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 effectcannot occur.

Warburg had already found that carbon monoxide inhibits the respiration of bakers yeast by combiningwith a component of the respiratory system of the cell [369]. During a visit to Warburg in the winterof 1927 1928, the English physiologist Archibald Hill10 told him about the light-sensitivity of thecarbon monoxidehaemoglobin 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 hisrespiratory 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, hemeasured the absorption spectrum of this Atmungsferment [373 375]. Furthermore, measuring theinhibition of respiration by his yeast in different mixtures of carbon monoxide and oxygen (replacingcarbon monoxide by nitrogen as a control), Warburg was able to calculate the relative affinity (K) of his

Atmungsfermentfor oxygen and carbon monoxide as a partition constant:

K =n

(1 n).

[CO]

[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 requiredvery 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 hehad to elaborate the theory for the quantitative interpretation of the measurements . . . It was this work for whichWarburg was awarded the Nobel Prize for Medicine and Physiology in 1931 [204, p. 27].

7 Atmung und Garung sind also durch eine chemische Reaktion verbunden, die ich nach ihrem Entdecker Pasteursche 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 mm

3 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 (18621931), Scottish physiologist, worked at Oxford with J. S. Haldane on air pollution caused by breathing.

He moved to Queens 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 Warburgs experiments on the action of light on the carbon monoxide inhibition of yeastrespiration. Reproduced with permission from [371, p. 81]. Dunkel= dark;hell= light

The Pasteur effect was studied further in many other kinds of cell. Using Warburgs methods toinvestigate the effects of carbon monoxide on the aerobic metabolism of several animal tissues, HansLaser12 confirmed some of Warburgs 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 anaerobicconditions; (c) the effect of carbon monoxide was reversed by light and he showed that, whereasrespiration 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 oxidationof a glycolytic enzyme by an electron carrier, such as a cytochrome [224]. As a development ofLipmanns view, in 1943, Vladimir Engelhardt13 and Nikolai Sakov14 established the major role of6-phosphofructokinase15 in producing the Pasteur effect [95]. Using fractionated muscle extract, theyinvestigated sensitivity to oxidation (by various redox dyes16) of certain enzymes of the glycolytic

12 Hans Laser (18991980), 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 (18941984) 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-Phosphofructokinasewas 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

E0 > +0.05 V, such as 2,6-dibromophenolindophenol or 2,6-dichlorophenolindophenol. E0is the approximate electrode potential, when

there are equal concentrations of both oxidized and reduced forms at pH 7. Relations of the oxidationreduction (redox) potential,

electromotive force and ionic concentration had been worked out in 1889 by Hermann Walther Nernst (18641941) [272].



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pathway and found only one of them to be sensitive, namely, 6-phosphofructokinase (PFK), whichcatalyses 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 highlysuggestive, was only of an indirect kind. But an impressive proof of the validity of the findings was obtained whenan 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, catalyticamounts 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 thePasteur effect under the most simplified conditions [94, pp. 910].

Since this work was finished during the middle of World War II, it was impracticable for the Russianauthors 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 authorswho 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 consistentwith those of Engelhardt and Sakov. First, Carl Cori was suggesting that PFK has a regulatory role inmuscle glycolysis, writing:

. . . hexosemonophosphate . . . a normal constituent of muscle . . . can increase considerably under certainexperimental conditions without any increase in the formation of lactic acid. This indicates that the reaction betweenfructose-6-phosphate and adenosinetriphosphate in intact muscle is a limiting factor as regards the rate at whichlactic acid is formed and carbohydrate is oxidized [70, p. 183].

Second, Joseph Melnicks17 findings, published in 1941 and 1942, accorded with the suggestion that thePasteur effect could be brought about by the action of cytochrome and cytochrome oxidase on PFK.The photochemical absorption spectra, obtained with bakers yeast, indicated that the three proteins,known as (a) Pasteur enzyme, (b) Atmungsfermentor (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 inhibitionby carbon monoxide and the photochemical absorption spectrum of the catalytic system involved in the Pasteurreaction [188, p. 268].

The allosteric effectors of 6-phosphofructokinase have been identified relatively recently, and theeffect of their inhibition is different for various organisms. Many allosteric inhibitors (more than 20,including cytochrome) for 6-phosphofructokinases have been foundin vitro. However, in vivo, the major

allosteric inhibitor of 6-phosphofructokinase is ATP and the major allosteric activators are fructose2,6-bisphosphate and AMP. The extent of activation and inhibition by these effectors differs betweenorganisms. Fructose 2,6-bisphosphate, first discovered in mammalian cells [357], is the main activator of6-phosphofructokinase18 in S. cerevisiae (see Figure 19, below) [191], (for review see [40]).

17 Joseph Lewis Melnick (19142001), American medical microbiologist, worked especially on enteroviruses at Yale University. He

became Professor of epidemiology in 1954 (Historical Register of Yale University, 19511968, 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 6-

phosphofructokinase-1, is a heterooctamer with 4 - and 4 -subunits [195], which are encoded by genes PFK1 (-subunit) and

PFK2 (-subunit) [67,159]. 6-Phosphofructokinase-1 phosphorylates D-fructose 6-phosphate to the glycolytic intermediate fructose 1,6-

bisphosphate, 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 4. Rates of oxidative respiration and non-oxidative fermentation by severalyeasts growing aerobically in 17 mM-D-glucose at pH 6.5 (temperature and growth phase

unspecified). Rates are in 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 butare 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 Dekens observations, as well as by some of Meyerhofs results described above, D-glucose almostcompletely represses the aerobic metabolism of many strains ofS. cerevisiae, even when oxygen is present.

Accordingly, such yeasts in the presence of glucose cannot show the Pasteur effect. Indeed, RosarioLagunas, 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 Bartley20

(Figure 2) and his colleagues stated that cells ofS. cerevisiae grown on glucose (at 50 mM or more) donot form mitochondria [289], the enzymes of the tricarboxylic acid cycle being repressed [287].

However, detecting mitochondria21 in anaerobically grown or glucose-repressed S. cerevisiae requiresspecial techniques for fixing and staining [72]. Since the 1960s, it has been accepted that this yeast whenmetabolizing anaerobically does have mitochondria in a smaller, somewhat elusive form [74] and thesehave sometimes been called promitochondria [285,311]. In the 1970s, Barbara Stevens, by means ofa remarkable electron micrographic study of serial thin sections and computer-aided three-dimensionalreconstructions, showed the volume of the promitochondria to occupy about 3% of the cell volume inglucose-repressed cells, and as much as 1012% in derepressed respiring cells [334].

Nonetheless, Lagunas and her colleagues have observed the Pasteur effect in Saccharomyces cerevisiaein resting (non-growing) cells [210,211], the resting condition being obtained by depriving the yeast of asource of nitrogen. When growing, the cells respired only 320% of the sugar they catabolized; whereasresting cells respired as much as 25100%. Accordingly, it became practicable to detect the Pasteur effectin such resting cells. Lagunas and her colleagues attributed this reduced rate of fermentation (>10% ofthat 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 (19161994), 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], Lagunascommenting further:

S. cerevisiae shows physiological characteristics very different from those often reported even in good textbooksof 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, Pasteurs finding is undoubtedly correct, namely, that the increase in cell mass anaerobicallyis 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, letalone 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 theseparticular yeasts may explain his findings.

First, the lower growth yield anaerobically was probably because these yeasts are unable to synthesizeergosterol and unsaturated fatty acids in the absence of oxygen, as Arthur Andreasen22 and TheodoreStier23 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 (19031991), 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 firstphase (about 06 h) is characterized by production of ethanol which, after the disappearance of glucose, is used as thecarbon source for growth (from [186]). Reprinted from Advances in Applied Microbiology 28, G. Kappeli, Regulation ofcarbon metabolism inSaccharomyces cerevisiaeand related yeasts: 181209, copyright 1986, with permission from Elsevier

(Figure 3) may be the underlying reason for the higher yield of biomass when oxygen is present. In phase1, 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:

C6H12O6 2EtOH + 2CO2, G = 235 kJ[205]

is much less than that for the aerobic oxidation of D-glucose, given by:

C6H12O6+ 6O2 6CO2+ 6H2O, G = 2873 kJ[205]

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]) andacetate (reviewed in [277]), generates an environment in which yeasts have an advantage, as they aregenerally more acid- and ethanol-tolerant than most bacteria. Hence, where there are high concentrationsof sugar, such as in rotting figs or grapes, these relatively slow-growing eukaryotic microbes can competesuccessfully with most (fast-growing) prokaryotes.

The Custers effect

In 1940, when working in Albert Kluyvers (Figure 4) laboratory in Delft, Mathieu Custers24 studiedyeasts of the genera Dekkera and Brettanomyces, which are important in the brewing of the rather acidBelgian lambic beer [147]. In contrast to the Pasteur effect, Custers described how these yeasts fermentD-glucose to ethanol fasterunder aerobic conditions than anaerobically [73]. He also reported that theyproduce 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 doctors 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 theCusters effect in 1966 [313].

Measuring respiratory exchanges with Warburg manometers, Scheffers found a marked Custers effect inDekkera 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 thesuspensions of this yeast. These additives included acetone, ether, acetaldehyde, acetone, pyruvic acid,formaldehyde, 3-hydroxy-2-butanone (acetoin25), 1,3-dihydroxyacetone, butanone (methyl ethyl ketone)and -oxoglutaric acid. He wrote:

The results suggest an action of the carbonyl compounds as H-acceptors in enzymatic dehydrogenation . . .Oxidizedcoenzyme I (DPN) [NAD+] enhances anaerobic fermentation to an extent depending on its concentration . . . it istentatively suggested that the inhibition of the start of fermentation inBr. 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 ableto 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 underanaerobic conditions (Figure 5) [313]. He and his colleagues published additional evidence that glycolysisis slowed by lowering the concentration of NAD+ (Figure 6) [50]. This is because production of aceticacid involves reduction26 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+ CH3 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: , inaerobic conditions + or exogenous 103 M 3-hydroxy-2-butanone (acetoin); , in anaerobic conditions; , in anaerobicconditions + 103 M3-hydroxy-2-butanone; , with 0.12% oxygen

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

The Kluyver effect

Kluyvers observations

In 1940, Kluyver and Custers reported that although Candida (Torulopsis) utilis can ferment D-glucoseanaerobically to ethanol and carbon dioxide, this yeast can (unlike Saccharomyces cerevisiae) utilizemaltose aerobically only. Thus they confirmed earlier reports that certain yeasts were able to use the



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Table 5.Abilities ofCandida utilis and Saccharomyces cerevisiae to utilizeD-glucose and maltose

Candida utilis Saccharomyces

cerevisiae

aerobic growth + +

D-glucose

fermentation + +

aerobic growth + +

maltose

fermentation

+

component hexoses of certain disaccharides anaerobically, yet could use those disaccharides aerobicallyonly [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 maltosecatabolism is:

maltose + H2O-glucosidase 2 D-glucose

why doesnt Candida utilis ferment maltose? Kluyver and Custers reasoned that the organism is ableto 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 thenineteenth century, had firmly established for yeasts that oligosaccharides are always hydrolysed beforethey are fermented27 ( [117], and see [28]). Hence, the inability ofC. utilis to ferment glucose was noteasy to interpret.

Indeed, Kluyver and Custers found no lack of -glucosidase activity in a strain ofKluyveromycesthermotolerans (Torulopsis dattila), which gave the Kluyver effect with maltose. Working in the late1930s, they suggested that the effect was caused by anaerobic conditions reversibly inactivating someglycoside hydrolases, such as -glucosidase [194, p. 159]. On 10 May 1940, the German army invadedHolland, so that Kluyvers research was severely interrupted for several years [185] and it was not untilthe 1950s that an alternative explanation became available; namely, inactivation of the mechanism oftransport across the plasma membrane. Such an explanation became feasible after Jacques Monod andhis 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 wereinterpreted 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 othercontexts, there were reports that certain yeasts required oxygen for the transport of sugars into theircells. For example, (a) a non-fermenting yeast, Rhodosporidium toruloides, was found to transport D-glucose actively under aerobic conditions, but not to take up that sugar anaerobically [202], and (b) a

27 In 1922, Richard Willstatter (18721942) and Gertrud Oppenheimer (18931948) had disputed Fischers 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 ofSaccharomyces pastorianus was shown to have a much reduced rate ofmaltose uptake compared with the wild-type [310].

Observations of Sims and Barnett

However, it was not until the late 1970s that Tony Sims28 and Barnett began investigating the physiologyof the Kluyver effect in yeasts [324]. Basing their information on a survey by taxonomists [231], they listedthe 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 Pasteureffect. Table 6 gives examples from these authors list and illustrates the finding that there was noobvious pattern of occurrence of the Kluyver effect; on the contrary, there was striking individualityamong yeasts in their response to each substrate.

Sims and Barnett extended the notion of the Kluyver effect to the utilization of D-galactose. The routeby 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 thecatabolism 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 acarbon dioxide electrode to measure CO2 output under both aerobic and anaerobic conditions. A nine-fold increase in the rate of CO2 output occurred only a few seconds after admitting air into an anaerobicsuspension 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 certainglycosides 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 + ? ? ? K K ? ? + ?

polymorphus

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,

,-trehalose; LAC, lactose; MEL, melibiose; MLZ, melezitose; MeG, methyl -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 h1 might both be registered as +. The results come from [29].

28 Anthony Peter Sims (19331990), 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 Candidautilis (NCYC 737). A suspension (0.4 mg dry wt/ml) ofC. utilis was carbon-starved aerobically for 2 hours in Difco yeastnitrogen 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 forabout 5 s () and the recording was continued; (ii) endogenous anaerobic CO 2 was recorded; 5 mol maltose (MAL )were added at about 2 minutes and air was admitted at about 3 minutes (O2 ), the yeast then again made anaerobic andfurther 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 processesinvolving induction or derepression, for which enzymic (or carrier) synthesis is essential (see [101]).Moreover, with C. utilis, which shows the Kluyver effect for the -glucoside, cellobiose,29 there was no

loss of-glucosidase activity associated with a change from aerobic to anaerobic conditions [324].

29 Whereas maltose (4-O--D-glucopyranosyl-D-glucopyranose) is an -linked glucose-glucose disaccharide, cellobiose (4-O--D-

glucopyranosyl-D-glucopyranose) is the same, but -linked:



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Since inactivity of the hydrolases did not appear to explain the Kluyver effect, it seemed worthinvestigating whether the carriers which take sugars into the cells might be deactivated, as had beensuggested previously [16,119]. The crude results from tests by taxonomists also indicated that transportmight well be an important factor. For those oligosaccharides which are mostly hydrolysed in thecytosol30 (Table 7), 70100% of the yeasts showed the Kluyver effect. On the other hand, for thoseusually hydrolysed outside the plasma membrane, the corresponding figure was


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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 cytosoland others hydrolysed externally to the plasma membrane. Results of ataxonomic survey; the list includes only those yeasts for which all strainsare able to (a) grow aerobically on the specified oligosaccharide and(b) ferment D-glucose. Data from [29]

Oligosaccharides Percentage of species

showing Kluyver effect

Usually hydrolysed in cytosol

melezitose 98

lactose 92

methyl-D-glucopyranoside 87

cellobiose 86

maltose 73

,-trehalose 54

Usually hydrolysed externally

sucrose 33

raffinose 24

melibiose 14

Entry of maltose into Candida utilis, too, was much slower anaerobically than aerobically. In furtherwork, on the unregulated maltose uptake of a mutant31 ofSaccharomyces cerevisiae, Barnett and Simsfound that the active transport of exogenous maltose ceases on switching from aerobic to anaerobic

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



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Table 9. Rates of uptake of D-[1-3H]fucose by galac-tose-grown Kluyveromyces thermotolerans under aerobic

and anaerobic conditions. Note: the inhibition of uptakeby D-galactose is consistent with the entry of D-fucose bytheD-galactose carrier. (From [324])

Condition Inhibitor Rate of uptake

nmol min1 (mg dry wt)1

Aerobic none 20.7

Anaerobic none 4.8

Aerobic D-galactose 1.28

Anaerobic D-galactose 0.84

Figure 11. The ability of a mutant strain of Saccharomyces cerevisiae, to concentrate exogenously-supplied maltose.

, Aerobic uptake; , anaerobic uptake; - - - - , equilibrium conditions, when exogenous and endogenous maltoseconcentrations are the same. Reproduced from Barnett and Sims 1982 [32]. The mutant, which was defective in glucoserepression, had uncontrolled uptake of maltose [98]

conditions so that, consequently, the yeast did not concentrate maltose anaerobically (Figure 11) [32].They extended their study of the requirement of oxygen for the active transport of sugars into otheryeasts, using strains of Kluyveromyces marxianus and Debaryomyces polymorphus. Experiments withthe non-metabolizable analogue of lactose, TMG32 (methyl 1-thio--D-galactopyranoside), showed that

these yeasts, too, required an oxygen supply for the active transport of lactose, which Barbara Schulzand Milan Hofer later confirmed for D. polymorphus [321].

Although Barnett and Sims found that active transport ceases under anaerobic conditions, facilitateddiffusion,33 by which the glycosides can also enter the cells, seemed to be unaffected. Hence, theyconcluded that the control mechanism underlying the Kluyver effect (a) probably also acts at a laterstage of catabolism, such as in the pathway from pyruvate to ethanol (Figure 12), and (b) is not mediatedby the slower processes involving induction or repression [32].

32 TMG (methyl 1-thio--D-galactopyranoside) was used by Adam Kepes for studying the kinetics of -galactoside transport into

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) inSaccharomyces cerevisiae and Candida utilis to be associated with the rate of catabolic flux in theanaerobic utilization (fermentation) of D-glucose [358]. Observations on six species of yeast by Sims andBarnett were consistent with these findings [325]. Five of these yeasts utilized one or more disaccharidesaerobically, but not anaerobically, although all used D-glucose anaerobically, that is, all five showed theKluyver effect; but the sixth yeast, S. cerevisiae, did not do so. When grown on a glycoside with whichit showed the Kluyver effect, each yeast had much less pyruvate decarboxylase activity than when grownon a glycoside with which it did not give the effect (exemplified in Table 10). There was no consistentcorresponding 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 pyruvatedecarboxylase, as described later for Kluyveromyces lactis [37], the enzyme being activated by itssubstrate, pyruvate [39,174,314,324,335].

(c) While switching to anaerobic conditions activates pyruvate decarboxylase, transport is greatly sloweddown by a reduction in the supply of ATP, so pyruvate decarboxylase activation fails because ofreduced glycolytic flux.

Experiments of Jack Pronk and his colleagues

Although some later work on maltose catabolism by Candida utilis, published by Jack Pronk and hiscolleagues at Delft in 1994, gave support to the notion that transport limitation is a factor in the Kluyvereffect, their findings with pyruvate decarboxylase conflicted with the idea that inactivation of that enzymewas also a factor [378]. They found that pyruvate decarboxylase activities ofC. utilis grown on maltose inoxygen-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 -glucosidases of Debaryomyces polymorphus have Km = 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 ofcarbon: expressed as nmol substrate catalysed min1(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

Saccharomyces

cerevisiae

D-glucose 1.62 0.57

maltose 0.40 0.64 1.01

a Yeasts grown on maltose were tested for -glucosidase activity. C. viswanathii grown on cellobiose was tested for-glucosidase activity.b C. viswanathiigave the Kluyver effect with cellobiose.

In order to test the hypothesis that yeasts, which show the Kluyver effect for sucrose, hydrolyseit 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 ofthe 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 ofKluyveromyces lactiswhich did not show the Kluyver effect from strains that already did so. Although 40 000 colonies ofmutagenized cells grown aerobically on lactose plates were replica-plated onto maltose, cellobiose or,-trehalose (all substrates giving the Kluyver effect with these yeasts), none of the colonies was ableto grow anaerobically on these sugars [100].

However, certain mutants failed to grow with glycerol. These fds mutants were totally aerobic anddepended entirely on anaerobic fermentation. However, they were not respiratory-deficient and, hence,were similar phenotypically to the glucose derepression mutant snf1 ofSaccharomyces cerevisiae (seebelow). When these mutants were tested against substrates that gave the Kluyver effect, none was utilizedaerobically. Poisoning respiration with KCN immediately prevented uptake of these substrates and ledto an instant decrease in the concentration of D-glucose 6-phosphate. Adding glucose to these poisonedcells 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,alpha-trehalose 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 ofgal2 mutants of Saccharomyces cerevisiae to use D-galactose anaerobically, although they will growon 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 galactoseanaerobically.

Results of some genetic experiments with Kluyveromyces lactis also give credence to the theory thatloss of the supply of metabolic energy, necessary for transport, has a r ole in producing the Kluyver effect.Paola Goffrini and her colleagues in Parma have been studying the curious case of the Kluyver effect with



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Figure 13.Raffinose

the trisaccharide, raffinose (O--D-galactopyranosyl-(1 6)--D-glucopyranosyl -D-fructofuranoside,Figure 13). This case is curious, because raffinose is usually hydrolysed outside the plasma membraneby invertase (cf. Figure 9) to produce melibiose and D-fructose. NowK. lactis does not utilize melibiose[29] and the fructose might be expected to be transported into the cells by a hexose carrier, as describedfor 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 theeffect. However, Goffrini and her colleagues report overcoming the effect in this yeast by introducingsugar 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 ofcertain 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 aerobicmetabolism described for Rhodosporidium toruloides [202], mentioned above, can be compared tomechanisms underlying the Kluyver effect. In any case, a study of both phenomena could well assistthe progress towards an understanding of the transport of sugars into yeasts. Furthermore, Pronk and hiscolleagues 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 andgrowth-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 differencesbetween these two phenomena, and so some explanation is given here of this effect and its history. In the1920s, following up Warburgs findings that certain tumour tissues have a higher rate of glycolysisthan normal cells [368], Herbert Crabtree35 studied the respiration of tumour cells and found that

35 Herbert Grace Crabtree(18921966), 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 Crabtreeeffect 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. Ifefficient glucose fermentation produces a high concentration of ATP in the cytoplasm, importing ofADP into the mitochondria is prevented, and the consequent depletion of ADP leads to a lower rate ofrespiration.

This, however, does not explain why 2-deoxy-D-glucose (2DG) produces a Crabtree effect [396]. In1958, Kenneth Ibsen and his colleagues [176] showed that the level of ATP decreases almost immediatelyafter 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 concludedthat the level of cytoplasmic ATP is overcome by a disproportionate reaction in the mitochondria of2ADP ATP + ADP, ADP being exported into the cytoplasm. This export decreases the concentrationof 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 addingglucose, and a long-term Crabtree effect, which occurred after 2030 minutes. The short-term effect wasexplained by an excess of ATP within the mitochondria and the long-term effect by reduced import ofADP into the mitochondria. Both result in depleted mitochondrial ADP.

Glucose repression in yeasts

The eccentric behaviour ofSaccharomyces cerevisiae, when supplied with D-glucose, has already beenmentioned in this series [31]: even in air, most of the pyruvate formed by glycolysis is channelled toethanol, rather than into the tricarboxylic acid cycle (Figure 14) and, accordingly, the yeasts respiratoryactivity 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 Crabtreeeffect after Crabtrees findings [78].

However, the physiological reasons for the lower rate of respiration after adding glucose are completelydifferent in yeast and tumour cells. Whereas, as described above, the respiratory decrease in tumourcells depends solely on metabolic changes (ADP depletion), the corresponding respiratory decrease inyeast cells is caused by the repression of the structural genes responsible for synthesizing respiratoryenzymes [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 termedit glucose effect [111]. Later, Boris Magasanik used the term catabolite repression instead [240]: in1961, 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 whichsuggests 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 aresynthesized at low rates or not at all. This phenomenon is known as carbon catabolite repression, or simply cataboliterepression, 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 glucoserepression, to stress that other sugars, such as galactose or maltose, are able to affect the synthesis of enzymesrepressed by glucose [129, p. 334].

In 1942, Epps and Gale had described their glucose effect as follows: the presence of glucose inthe medium during the growth ofEscherichia coli suppresses the formation of certain enzymes [111].



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(A)

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

Solomon Spiegelman and John Reiner in 1947, and Wilbur Swanson36 and Charles Clifton37 in 1948,reported a similar finding for Saccharomyces cerevisiae (or S. pastorianus) [331,342]. In their excellentpaper, Spiegelman and Reiner carefully examined the galactose metabolizing pathway, which they referredto as galactozymase (see [25]). They observed that a yeast pre-grown with galactose, and thereaftertransferred to a glucose-containing medium, lost its galactozymase activity; but this loss was preventedby adding azide38 (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 growthmedium 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 (enzymeactivity 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 theydid 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 (19041976), 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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Figure 16. The tricarboxylic acid and glyoxylate cycles. Reproduced from [197], courtesy of H. L. Kornberg and bypermission of Elsevier

culture on 56 mM 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 1964and 1965) which described a major step towards understanding glucose repression in Saccharomycescerevisiae. Sugars in the medium for growing this yeast aerobically caused an anaerobic type ofmetabolism as measured by ethanol production, D-glucose being much more effective in this respectthan was D-galactose. This glucose repression affected enzymes of the tricarboxylic acid cycle, bothglucose and galactose repressing the key enzymes of the glyoxylate cycle39 (Figure 16) almost completely.Furthermore, glutamate dehydrogenase was more than 50 times more active in S. cerevisiae when it wasgrown on pyruvate than on D-glucose [287]. In addition, Bartley and his colleagues found no mitochondrialstructures in yeast grown aerobically on glucose but, with the removal of glucose, mitochondria reappearedas the yeasts ability to respire acetate returned [288,289]. And in 1971, Alberto Sols and his colleagueswrote:

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. Gorts, 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, andis currently under study in several laboratories [330, p. 301].

Many kinds of enzyme in yeasts have been found to be subject to glucose repression. Theseinclude 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]).

Oxaloacetate

Malate

Acetate Glyoxylate Succinate

isoCitrate

Citrate

Acetate

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 Escherichiacoli differed from that in yeasts. Research on E. coli had shown D-glucose to lower the levels ofcAMP40, which nucleotide is necessary for the transcription of genes sensitive to carbon cataboliterepression [177,353]. However, evidence was accumulating that this was not true of yeasts [241]. Addingexogenous cAMP to strains of Saccharomyces cerevisiae that were permeable to it did not preventthe repression of galactokinase [246] and levels of cAMP were at least twice as high in repressedS. 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 completelyfrom that in S. cerevisiae. In E. coli, binding of cAMP to the cAMP receptor protein (CRP or CAP 41) isnecessary for the transcription of glucose repressible enzymes [93,323,400]. By contrast, there is no CRPhomologous protein inS. cerevisiae and, unlikeE. coli, there is a transient increase in cAMP concentrationin S. cerevisiae within 2 minutes of adding glucose [248,291,356]. Indeed, there is no evidence that thelevel 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 todecide which name should be used for each gene. Genetic convention is to prefer the name used in thefirst description of a mutant. However, many mutants which proved to be synonymous were isolatedindependently. Often their allelism was demonstrated much later than their original description, in manycases only after their respective wild-types had been isolated and sequenced. Accordingly, the chosenname of each gene could be that used when it was first sequenced.

Mark Johnston has suggested that, like the nomenclature of mitochondrial proteins, the glucose-repression community should decide on new names, each of which would refer to the genes function.Although this idea is attractive, this historical survey is no place to generate further confusion with yetmore names. Accordingly, in order to help follow the complexities of the molecular-genetic control ofglucose repression, the large number of pleiotropic genes involved and their synonyms are summarizedin 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, -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 onlycharacterized it biochemically [137,261]. Mutantflk1 was found later to be allelic totup1, whose functionis 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

(aconitase)

S. cerevisiae 51 Comparing growth in 50 mM D-glucose with

that in 44 m Mpyruvate [287]

ALCOHOL DEHYDROGENASE P. anomala 39 Grown in 167 mM D-glucose; then incubatedS. cerevisiae 91 for 4 h in (a) 56 mM 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]

-FRUCTOFURANOSIDASE

(invertase or inulinase)

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

ofD-glucose, from 250 mMto 3 M [85]

S. pastorianus 91 Comparing growth in 111 mM D-glucose withthat in 17 m M D-glucose [310]

FRUCTOSE BISPHOSPHATASE C. salmanticensis >98 Comparing growth in 111 mM D-glucose withD. carsonii 69 that in 0.43 M ethanol [130]D. hansenii 62P. anomala 82P. membranifaciens 90Rh. glutinis 86Rh. minuta 52Rh. mucilaginosa 89Rhs. toruloides 76Sp. pararoseus 95S. cerevisiae >98

-GALACTOSIDASE K. marxianus >99 Comparing growth in 111 mM D-glucose with


-GLUCOSIDASE K. marxianus

K. dobzhanskii


that in 0.1 m M D-glucose [239]

GLUTAMATE DEHYDROGENASE S. cerevisiae 50 Comparing growth in 50 mM D-glucose with


ISOCITRATE DEHYDROGENASE

(NAD+)



30 Grown in 167 mM D-glucose; then incubated

for 4 h in (a) 56 mM D-glucose or (b) 61 mM


ISOCITRATE DEHYDROGENASE

(NADP+)



In 1975 Michael Ciriacy44 (Figure 17) devised an electrophoretic system by which he could distinguishthree isoenzymes of alcohol dehydrogenase: alcohol dehydrogenase I (Adh1p45), alcohol dehydrogenaseII (Adh2p) and mitochondrial alcohol dehydrogenase (Adh3p). He showed that Adh1p is mainly present

44 Michael Ciriacy (19471996), 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 ofSaccharomyces

cerevisiae genetically. He was in Fritz Zimmermanns laboratory at Darmstadt from 1977 to 1981, when he became a professor at the

Institute of Microbiology at the University of Dusseldorf [160].45 Abbreviations used for proteins, for which each gene is responsible, are written as the abbreviation of genes 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 P. anomala 94 Grown in 167 mM D-glucose; then incubatedRh. glutinis 99 for 4 h in (a) 56 mM D-glucose or (b) 61 mM

S. cerevisiae 99 NaAc; (a) compared with (b) [88]


that in 61 m MNaAc [389]

MALATE DEHYDROGENASE P. anomala 3 Grown in 167 mM D-glucose; then incubated

for 4 h in (a) 56 mM D-glucose or (b) 61 mM


S. cerevisiae 44



MALATE SYNTHASE P. anomala 94 Grown in 167 mM D-glucose; then incubated

Rh. glutinis 99 for 4 h in (a) 56 mM D-glucose or (b) 61 mM

S. cerevisiae 95 NaAc; (a) compared with (b) [88]


44 m Mpyruvate [287]



OLIGO-1,6-GLUCOSIDASE

(isomaltase)

S. pastorianus 87 Comparing growth in 111 mM D-glucose with


PHOSPHOENOLPYRUVATE C. pelliculosa 67 Comparing growth in 111 mM D-glucose with

CARBOXYKINASE Cr. humicola 55 that in 0.43 M ethanol [127]

P. anomala 50

Rh. glutinis 5

Rh. mucilaginosa 10

S. cerevisiae >65S. pastorianus 975

Z. fermentati 63

Z. rouxii 79

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 Adh2pis subject to glucose repression [60,61]. He also described a mutation of the ADH2 promoter46 whichmade alcohol dehydrogenase II47 insensitive to glucose repression [62]. Molecular analysis showed thatthis insensitivity was caused by a promoter insertion of the yeast transposon Ty1 [384].

The first pleiotropic48 mutants of glucose repression,cat1 andcat2, were isolated by Fritz Zimmermann

in 1977 [398] by screening for mutants that could grow on glucose, but not on ethanol, as thecarbon source. The cat1 mutants failed to derepress various enzymes: -glucosidase, invertase, andalso gluconeogenic and respiratory enzymes; hence, these mutants did not grow with ethanol, maltose orsucrose as a sole source of carbon.

That same year, Ciriacy, who later joined Zimmermanns laboratory, established a system for selectingmutants in which there was no glucose repression. Ciriacy used haploid mutants lacking a constitutivealcohol dehydrogenase49 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.47 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.



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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 derepressionof (a) enzymes of gluconeogenesis, (b) isocitrate lyase (of the glyoxylate cycle; see Figures 16 and 18)or (c) fructose bisphosphatase50 (Figure 19), that is, the strains carrying ccr1 could not synthesize theseenzymes, whether or not glucose was present. Ciriacys ccr1 mutant was allelic to Zimmermanns 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+ H2O D-fructose 6-bisphosphate+ ortho-phosphate. Fructose bispho-

sphatase 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 (Schuller and Entian, unpublished),isolated by Marian Carlson [48] (see Table 12).

Thecat1 mutation affects all the glucose-repressible enzymes and, as described below, later biochemicalanalysis has shown that cat1 encodes the most central element in the regulatory circuit of glucoserepression, a protein kinase, named Snf1-4-kinase (see below).

Zimmermanns selection system for mutants defective in glucose repression

A further advance in the genetic analysis of glucose repression was Zimmermanns development in1977 of a powerful system for selecting mutants which resisted glucose repression [399] (described inan earlier article in this series [25]). Working with Saccharomyces cerevisiae growing exponentially onglucose as carbon source, he plated this yeast on medium containing low concentrations (0.61.8 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 istoxic, 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].



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Table 12. Genetic and biochemical characterization of genes involved in glucose repression Asterisk indicates firstdescription

Gene Mutant isolation Mutant phenotypes Gene

sequence

Physiological role of wild-type protein

Subunits of the central Snf/Cat complex

CAT1 Zimmermann and

colleagues 1977

[398]

No growth on non-fermentable

carbon sources; no derepression of

-glucosidase, invertase,

gluconeogenic or glyoxylate cycle

enzymes

Zimmermann and colleagues 1977

Celenza and

Carlson

1986 [52]

Catalytic subunit of a Ser/Thr-specific

protein kinase complex, phosphorylates

activators and repressors involved in

glucose repression.

Celenza and Carlson 198

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Yeast metabolism