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A history of research on yeasts 9: regulation of sugar metabolism1

2005; Wiley; Volume: 22; Issue: 11 Linguagem: Inglês

10.1002/yea.1249

1097-0061

James A. Barnett, Karl‐Dieter Entian,

Genetic Neurodegenerative Diseases

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 catabolite repression, and glucose or catabolite inactivation (Table 1). What has been called the Crabtree effect in yeasts should, as will be discussed below, be called 'glucose repression'. Such regulatory effects 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 phenomena described, as well as an exposition of their physiological rôles in Saccharomyces cerevisiae and, as far as is known, in other yeasts too. An account is also given of the history of research on the inter-regulation of glycolysis and gluconeogenesis.2 The story of studying these processes, like many aspects of microbiology, began with the work of Louis Pasteur3 who, in 1861, described how the growth of yeast per gram of sugar consumed was much greater under aerobic than anaerobic conditions [279]. 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 1937 [363, 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, and announce boldly, not 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-that-they-could-all-be-right class that we must be wary of (Dean Burk4 1939 [46, p. 421]). As considerable confusion exists in the current literature as to the real nature of the Pasteur effect, it is necessary to explain Pasteur's original conceptions and to describe his experimental results on the effect of oxygen on carbohydrate catabolism (Kendal Dixon5 1937 [84, p. 432]). 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 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]). Respiration and fermentation are thus connected by a chemical reaction, which I call the 'Pasteur reaction' after its discoverer.7 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 [256]). This finding of Meyerhof's has since been reported many times for strains of Saccharomyces cerevisiae (e.g. [340]): 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. Results of one of Warburg's experiments on the action of light on the carbon monoxide inhibition of yeast respiration. Reproduced with permission from [369, p. 81]. Dunkel = dark; hell = light … 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 [202, p. 27]. 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 [211]. 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]. … 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]. 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 [186, 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 [355], is the main activator of 6-phosphofructokinase18 in S. cerevisiae (see Figure 19, below) [189], (for review see [40]). Table 3 shows the chronological sequence of some of the work on the Pasteur effect. In 1966 R. H. De Deken19 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 [225] and Kluyveromyces lactis [306] 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 [225] and Schwanniomyces occidentalis [284]. 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 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 [206]. Furthermore, Walter Bartley20 (Figure 2) and his colleagues stated that cells of S. cerevisiae grown on glucose (at 50 mM or more) do not form mitochondria [287], the enzymes of the tricarboxylic acid cycle being repressed [285]. Walter Bartley. Photo courtesy of Joan Brown However, detecting mitochondria21 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' [283] [309]. 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 [332]. 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 [207, 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 Andreasen22 and Theodore Stier23 found in the 1950s [4] [5]. Second, the biphasic (or diauxic) growth of S. cerevisiae on glucose (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. 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 [184]). 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 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 [278]) and acetate (reviewed in [275]), 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. In 1940, when working in Albert Kluyver's (Figure 4) laboratory in Delft, Mathieu Custers24 studied yeasts of the genera Dekkera and Brettanomyces, which are important in the brewing of the rather acid Belgian lambic beer [146]. 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 [380]). Lex Scheffers and his colleagues confirmed the existence of this effect in a number of strains of Brettanomyces and Dekkera [380] and renamed it the 'Custers effect' in 1966 [311]. Albert Jan Kluyver. Photo courtesy of C. T. Kluyver 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 [310, p. 41]. Fermentation of D-glucose by Dekkera bruxellensis (CBS 1943). Results of Scheffers, published in 1966. Reproduced from [311], courtesy W. A. Scheffers and by permission of Nature Publishing Group. Symbols: ●, in aerobic conditions + or − exogenous10−3 M 3-hydroxy-2-butanone (acetoin); ○, in anaerobic conditions; □, in anaerobic conditions + 10−3 M 3-hydroxy-2-butanone; ▵, with 0.12% oxygen 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 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 component hexoses of certain disaccharides anaerobically, yet could use those disaccharides aerobically only [192]. Thirty-eight years later, this phenomenon was named the Kluyver effect [322]. 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 [192, p. 159]. On 10 May 1940, the German army invaded Holland, so that Kluyver's research was severely interrupted for several years [183] 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. [298], 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 D-glucose actively under aerobic conditions, but not to take up that sugar anaerobically [200], and (b) a respiratory-deficient mutant of Saccharomyces pastorianus was shown to have a much reduced rate of maltose uptake compared with the wild-type [308]. However, it was not until the late 1970s that Tony Sims28 and Barnett began investigating the physiology of the Kluyver effect in yeasts [322]. Basing their information on a survey by taxonomists [229], 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 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. Routes of galactose and glucose catabolism (Leloir pathway simplified) These workers studied yeasts which gave this effect with maltose, cellobiose and D-galactose, using a carbon 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 anaerobic suspension of maltose-grown Candida utilis and was immediately linear (Figure 8). The rapidity of the 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 [322]. 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 CO2 was recorded; 5 µmol 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 [322] Since inactivity of the hydrolases did not appear to explain the Kluyver effect, it 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 well be an important factor. For those oligosaccharides which are mostly hydrolysed in the cytosol30 (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% [322] (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-glucosidase (Figure 9; for review, see [18]). Accordingly, Sims and Barnett measured the rates of entry of sugars into the cells. External hydrolysis of sucrose by invertase and internal hydrolysis by α-glucosidase Results of experiments with Kluyver's Kluyveromyces thermotolerans (Torulopsis dattila) were consistent with the transport of D-[1-3H]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), illustrating the important fact that the Kluyver effect occurs only with certain transport systems in any given yeast. Structures of D-galactose and D-fucose Entry of maltose into Candida utilis, too, was much slower anaerobically than aerobically. In further work, on the 'unregulated' maltose uptake of a mutant31 of Saccharomyces cerevisiae, Barnett and Sims found that the active transport of exogenous maltose ceases on switching from aerobic to anaerobic 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 other yeasts, using strains of Kluyveromyces marxianus and Debaryomyces polymorphus. Experiments with the non-metabolizable analogue of lactose, TMG32 (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 later confirmed for D. polymorphus [319]. The ability of a mutant strain of Saccharomyces cerevisiae, to concentrate exogenously-supplied maltose. ○, Aerobic uptake; ●, anaerobic uptake; ----, equilibrium conditions, when exogenous and endogenous 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] Although Barnett and Sims found that active transport ceases under anaerobic conditions, facilitated diffusion,33 by which the glycosides can also enter the cells, seemed to be unaffected. Hence, they concluded that the control mechanism underlying the Kluyver effect (a) probably also acts at a 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]. 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 [356]. Observations on six species of yeast by Sims and Barnett were consistent with these findings [323]. 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. Glycolytic flux may be low as a result of a combination of: The change from active transport to facilitated diffusion, which leads to a low concentration of glycoside in the cytosol and; The low affinity of the glycosidase for its substrate.34 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] [173] [312] [322] [333]. 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. 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 [376]. 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 [377]. 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 [182, p. 1567]. 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]. 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 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. 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 [138a, p. 427]. Raff inose 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]. 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 [366], Herbert Crabtree35 studied the respiration of tumour cells and found that adding glucose decreased the respiration rate [71] [174]. 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] [290] 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 [394]. In 1958, Kenneth Ibsen and his colleagues [175] 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 [162], 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. 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 t