A history of research on yeasts 13. Active transport and the uptake of various metabolites 1
2008; Wiley; Volume: 25; Issue: 10 Linguagem: Inglês
10.1002/yea.1630
ISSN1097-0061
Autores Tópico(s)Biotin and Related Studies
ResumoThe present article is particularly concerned with solutes crossing membranes against their own gradient of electrochemical potential; that is, transport which involves expending metabolic energy. This follows on from history article No. 11106, which dealt mainly with the uptake of solutes by facilitated diffusion, the energy for which depends on a concentration gradient. Research in the 1920s and 1930s showed salts to be taken up by roots of plants against a concentration gradient and that this uptake is associated with metabolic activity, as indicated by related increases in CO2 production by the plants229, 231, 293. Evidence of active transport of ions was also found in various animals, such as that for potassium into eggs of Oryzias latipes, the Japanese medaka fish168, or for the ability of frogs in need of salt to take up sodium chloride from their watery environment, even when that water contains only 10 µ M NaCl206. However, in 1943, Hugh Davson3 commented: Until something more is known about the driving force which determines the migration of an ion in these systems, it is not very profitable to discuss this migration from a simple permeability point of view84p.176. In the 1940s and 1950s, the active transport of ions, amino acids and glycosides was also described for bacteria and for human erythrocytes (Table 1), the study of active transport into yeasts taking off in the mid-1950s. The finding in 1956, by Jacques Monod4 and his colleagues at the Institut Pasteur in Paris, that Escherichia coli concentrates β-D-galactosides above the exogenous levels303, was followed the next year by a paper from J. J. Robertson and Orin Halvorson of Michigan University, describing the concentrating of methyl α-D-glucoside by cells of Saccharomyces cerevisiae307, its transport being sensitive to 2,4-dinitrophenol.5 Furthermore, in 1963, using the non-metabolizable thioglycoside, [35S]ethyl 1-thio-α-D-glucopyranoside (TEG), Halvorson published more evidence that S. cerevisiae can take up and accumulate this compound actively273-275 (Figure 1). Meanwhile, G. Harris and C. Thomson, of the Brewing Industry Research Foundation in England, investigated the uptake of maltotriose, an important constituent of brewer's wort,6 by a strain of brewer's yeast (S. cerevisiae).7 They found that, when suspended in 25 µ M [14C]maltotriose: (a) the intracellular concentration of maltotriose was more than five times that in the suspending medium after incubation for 2 h; (b) uptake was inducible; and (c) the sugar flowed out of the cells when placed in medium free of maltotriose (Figure 2)154. These two authors also published evidence that maltose was concentrated by S. cerevisiae155. Time course of accumulation of ethyl 1-thio-α-D-glucopyranoside (TEG) in cells of Saccharomyces cerevisiae at 30 °C. Results of Okada and Halvorson published in 1964, reprinted with permission from Elsevier from274. Cells were grown in nutrient medium containing 0·1 M TEG, washed and suspended in the same nutrient medium with TEG replaced by 0·1 M acetate (pH 5·8). [35S]TEG was added at zero time and samples containing 1–2 mg yeast were filtered, washed and counted Uptake and release of [14C]maltotriose by a non-maltotriose-catabolizing brewing strain of Saccharomyces cerevisiae. The yeast was suspended in the labelled sugar at zero time. Yeast (a) adapted, (b) not adapted to maltotriose; (c) after resuspension in medium free of maltotriose. Results of Harris and Thompson published in 1960154 Also in the 1960s, Arnošt Kotyk and Milan Höfer, working in Prague, studied uptake of sugars by a non-fermenting (that is, entirely aerobic) yeast, Rhodotorula gracilis (Rhodosporidium toruloides). In particular, Kotyk and Höfer examined the transport of sugars which this yeast metabolized only slowly or not at all and found that the yeast concentrated those sugars within its cells193p. 40. These authors' most convincing result was with [14C]L-rhamnose,8 which was concentrated 90-fold under aerobic conditions202. The fact of active transport of metabolites into yeasts having now, by the 1960s, been well established experimentally, clearly the next stage was to investigate the mechanism by which substrates entered the cells: how was the uptake energized? In 1961, Peter Mitchell had suggested a corollary of his chemiosmotic concepts, namely, that the reason why translocation of galactose and β-galactosides into Escherichia coli was sensitive to 2,4-dinitrophenol was because the movement was coupled to the influx of protons across the plasma membrane [254,255;256, p. 148]. This influx would necessarily be balanced by expelling protons from the cytosol back into the suspending medium (Figures 3, 4). By the mid-1970s, proton symport was fairly well established as one of the means by which some substrates enter certain bacteria. However, in 1974, the American biochemist Franklin Harold wrote: Peter Mitchell's diagram, published in 1963, 'of cyclic coupling between the electron-translocation system, thought to be present in the plasma-membrane of Escherichia coli … and an H+-galactoside system which … may translocate galactosides into the cell'255. (Diagram reproduced, courtesy of Cambridge University Press, from: Mitchell PD. 1963. Molecule, group and electron translocation through natural membranes. Biochemical Society Symposium 22: 142–167.) Diagram of proton symport coupled to a proton pump, which is driven by hydrolysis of ATP. Cells and organelles, such as mitochondria, actively transport H+ ions across their limiting membranes. This activity leads to (a) negative potential inside (relative to the outside) and (b) a concentration gradient of H+ ions. The tendency for the H+ ions to flow back out through the membrane may be coupled to, and thus energize, the transport of substrates into the cell or organelle. S, solute, such as a sugar, is taken up with one or more equivalents of protons. Energy for the ejection of protons is supplied by the hydrolysis of ATP. (After a diagram by Eddy104) The molecular mechanism of transport, and thus the concrete meaning of the facile term 'carrier,' remains virtually unknown. The literature on membranes and energy transduction conveys a pervasive sense of uncertainty. The principles of classical biochemistry, so eminently successful in treating scalar phenomena, are no longer sufficient; a new doctrine, firmly rooted in the structured nature of biological membranes, is still struggling to emerge. The chemiosmotic hypothesis is currently the best candidate for the new paradigm; in some ways, it is the only one153pp. 297 and 308. Indeed, the concentration of certain amino acids and sugars by various mammalian and bacterial cells had been shown to depend on the coupling of transport to the flow of specific cations, such as Na+, K+, or H+152a., 256, 304. For yeasts, Alan Eddy and his colleagues published, in the 1960s and early 1970s, evidence of the rôle of H+ or K+ as co-substrates in the uptake of amino acids into Saccharomyces (carlsbergensis) pastorianus9 103, 105, 109; these cells, made ATP-deficient by adding 2-deoxy-D-glucose and antimycin,10 nonetheless concentrated exogenously-supplied amino acids, provided that there was a relatively high external concentration of H+ and a low concentration of K+. Some of these authors' recordings of proton uptake are reproduced in Figure 5. For several yeast species, certain sugars and inorganic phosphate were shown to enter the cells by proton symport71, 109, 329, 330, a stoicheiometric number of protons being taken up as co-substrates and, probably, by an independent process. This uptake was balanced by the expulsion of an equivalent number of K+ ions. A strain of Saccharomyces cerevisiae, supplied with exogenous glycine, could concentrate it 100–200-fold and adding glycine or L-phenylalanine to the cell suspension immediately stimulated the rate of uptake of protons two- or three-fold. Effects of amino acids on proton uptake by Saccharomyces pastorianus (carlsbergensis) in the presence or absence of 2-deoxy-D-glucose (2-deoxy-D-arabino-hexose, 2DG) or antimycin (Ant). The length of the bar lines shows the pH change produced by adding 1 µ M HCl. The yeast (10 mg/ml) was: (i) suspended in 5% (w/v) glucose at pH 4·5 for 45 min; (ii) washed and resuspended at 50 mg dry wt/5 ml of 5 mM Tris buffer at pH 4·5. Trace (a) shows the effect of adding glycine 1 µ M at time indicated by the arrow. For trace (b), 10 µg Ant and 17 mM 2DG were present throughout and 1 µ M glycine was added at the arrow. Trace (c) shows four successive additions of 0·5 µ M glycine were made before the inhibitors were added in above amounts and, finally, another 0·5 µ M glycine added. Trace (d): no inhibitors; amino acids added in the order DL-2-aminoisobutyric acid (Aib), L-phenylalanine, L-leucine, L-lysine, glycine, DL-2-aminoisobutyric acid (0·5 µ M in each case). (Reproduced with permission from: Eddy AA, Nowacki JA. 1971. Biochemical Journal 122: 701–711. © The Biochemical Society.) A previous article in this series (No. 11106) describes some of the earlier work on the uptake of monosaccharides into Saccharomyces cerevisiae. In the second half of the1960s, Kotyk had described three monosaccharide carriers200, 203. One carrier was reported to transport all of the monosaccharides tested. A second was more specific for sugars which are similar structurally to D-glucose; the more the sugar differed from D-glucose in the orientation of its hydroxyl groups, the lower the affinity of the carrier for that sugar (see [106, pp. 1045–1046]). Although glucose transport by Saccharomyces cerevisiae was at this time thought to be mediated by only two or three kinetically distinct carriers, by 1999 about 34 sugar carrier proteins had been identified. These carriers included four for maltose, two glucose sensors and about 17 hexose (Hxtp) carriers. Their designation has been based on the results of both sequencing and transport experiments. In 1987, Linda Bisson and her colleagues were the first to isolate mutants unable to take up glucose; these mutants were defective in gene SNF311 44. In 1999, Roman Wieczorke and his colleagues, by deleting all the HXT genes in one strain, made it practicable to characterize each gene by re-introducing them, one by one, back into that strain. Each of these genes, except HXT12, enabled the yeast to grow on glucose398, while a yeast in which all the hexose-carrier genes, HXT1 to HXT17, SNF3, RG2 and GAL2, were deleted could not transport or utilize glucose. Nearly all the hexose carriers were found to transport glucose, fructose, mannose or galactose. Table 2, which summarizes information about the hexose carriers, shows that most of the work on these carriers and their structural genes was done in the 1990s (for reviews, see42, 47, 49, 70, 209, 211, 283). A group of workers from three German universities has explained the rationale of some of these complexities: Transport of glucose across the plasma membrane into the cell is the first step of glucose metabolism. Saccharomyces cerevisiae can deal with extremely broad ranges of glucose concentrations and glucose can be metabolized effectively at concentrations from higher than 1·5 M down to micromolar concentrations395. This implies the presence of a highly complex and highly regulated glucose uptake system [57, p. 283]. Eckhard Boles and Cornelis Hollenberg have suggested that a carrier in S. cerevisiae with a low affinity for hexoses is important for wine making: The properties described for [the low-affinity glucose carrier] Hxt1p qualify it as an important glucose transporter under conditions of extremely high sugar concentrations … [as] in grape juice which … contains up to 1·5 M combined glucose and fructose [49, p. 93]. Hxt1–7p and Gal2p are the main hexose transporters of Saccharomyces cerevisiae, each acting by facilitated diffusion. Transcription of the HXT genes, which encode the seven Hxt carriers, is controlled both by glucose repression and induction. Glucose induction involves two glucose sensors, Snf3p and Rgt2p283. In the presence of glucose, the sensors send signals to the nucleus via several proteins, so effecting transcription of the hexose carrier genes. Even when over-produced, Snf3p and Rgt2p do not restore the ability to grow on glucose to HXT-deficient strains96, 280, so they act solely as sensors and, if they are also carriers, they transport glucose insufficiently fast to support growth, the sensors acting like receptors or having minor carrier activity which initiates a signal48. As described briefly in history article 11106, the first advances in studying the uptake of oligosaccharides by yeasts came in the 1950s and early 1960s. (a) In 1949 Alfred Gottschalk12 suggested that the rate of a substrate's entry into the cell might determine its rate of catabolism130, 131, thereby explaining such curious phenomena as the faster utilization of lactose than of its components, glucose and galactose (see405). (b) The careful and ingenious investigations of Alberto Sols and Gertrudis de la Fuente of glycoside utilization by several yeast species in the early 1960s showed that, although most glycosides entered the cells by means of carrier-mediated transport, some glycosides, such as sucrose and raffinose for Saccharomyces cerevisiae, were hydrolysed initially outside the plasma membrane91, 343. The findings, also in the 1960s, that glycosides are taken into yeast cells by active transport are described above and, in the 1970s, the ability to concentrate certain glycosides was found to depend on proton symport. Eddy and his colleagues showed that a strain of Saccharomyces cerevisiae (carlsbergensis) grown on maltose absorbed with it two to three equivalents of protons52, 53, 330. They found the uptake of protons was accelerated when the yeast was incubated with methyl α-D-glucopyranoside, turanose or sucrose (the latter, presumably in this instance, hydrolysed in the cytosol), but not when incubated with D-glucose, D-galactose or 2-deoxy-D-glucose, since all three sugars entered by facilitated diffusion. These authors also found another yeast, a strain of Kluyveromyces marxianus (Saccharomyces fragilis), absorbed extra protons in the presence of lactose, which it catabolizes. Much like the research on the uptake of sugars, the sequence of findings on the uptake of nitrogen compounds has been as follows: (i) various nitrogen sources are taken up by yeast cells; (ii) uptake involves saturation kinetics; (iii) mutants, exhibiting Mendelian genetics, are obtained with modified uptake characteristics; (iv) uptake requires metabolic energy and occurs in plasma membrane vesicles, in many cases independently of subsequent chemical reactions; (v) the relevant gene codes for a protein with the attributes of a membrane protein; (vi) (more recently) specific amino acid substitutions lead to subtle changes in the behaviour of the system; (vii) there are striking resemblances between yeast transport proteins and their counterparts in other organisms. One of the earliest papers on the uptake of amino acids by a yeast was that of Halvorson and his colleagues152 who, in 1955, published evidence that nitrogen-starved cells of Saccharomyces cerevisiae can concentrate externally-supplied arginine, glutamic acid or lysine up to 1000-fold (Figure 6). However, most amino acid carriers were discovered by a combination of biochemical and genetic methods, many by Marcelle Grenson13 (Figure 7) and her colleagues (Table 3). Uptake of amino acids by Saccharomyces cerevisiae; modified from a paper by Halvorson and his colleagues published in 1955152. Nitrogen-starved cells were aerated at 30 °C and exposed to various concentrations of amino acids for 5 min. After washing, the endogenous pools were estimated, using specific decarboxylases, by paper chromatography; organic nitrogen was estimated by the Kjeldahl method, by which the nitrogen is converted into ammonium sulphate, devised in 1883 by Johan Gustav Kjeldahl (1849–1900)192 Marcelle Grenson (photo courtesy of Bruno André) Several publications in the 1950s and 1960s reported the uptake by yeasts of various amino acids, exploiting competitive inhibitions between each of them to give kinetic evidence of wide specificity of the amino acid carrier(s) of Saccharomyces cerevisiae151, 152, 355, 356. Grenson, in particular, obtained many mutants which lacked the ability to transport a specific amino acid or certain structurally similar molecules, thus providing information, from genetic evidence, of the specificity of the amino acid carriers. Table 4 summarizes findings for amino acid transport genes of S. cerevisiae. The growth of Saccharomyces cerevisiae having been shown to be inhibited by canavanine (an analogue of arginine)14 349, in 1962 Grenson and her colleagues isolated mutants of Saccharomyces cerevisiae which were resistant to canavanine, as Adrian Srb15 had described previously350. Canavanine mutants of Escherichia coli had already been found unable to transport L-arginine325 and Grenson's mutants of Saccharomyces cerevisiae proved similarly deficient396. Also using mutants and competition experiments, Grenson later concluded that S. cerevisiae has a specific transport system for L-arginine because most other amino acids do not act as competitive inhibitors of its uptake148 and there is a specific carrier for L-lysine, which also enters by the L-arginine carrier137, another for L-methionine125 and yet a third for the dicarboxylic amino acids, aspartate, 2-aminoadipate and glutamate182. As well as possessing carriers for specific amino acids, in 1970 Grenson and her colleagues confirmed that Saccharomyces cerevisiae also has a general amino acid carrier (GAP, for 'general amino acid permease')147, as had been reported previously151, 356. They found this carrier had wide specificity, even taking into the cells D-amino acids, such as D-histidine, D-methionine and D-serine320. GAP is repressed when the yeast is grown with NH4+ as sole source of nitrogen147 and Grenson gave the name gap to a mutant which lacked GAP activity. The transport systems which Grenson, her colleagues and others described are summarized in Table 5, while Table 6 lists some characteristics of amino acid carriers of S. cerevisiae. The repressive effect of ammonia on the utilization of nitrogen compounds is analogous to the repressive effect of glucose on the uptake and catabolism of many carbon compounds, as discussed in a previous article in this history series [27, pp. 857–877]. In the 1980s, Grenson and others unravelled some of the complexities of the controls on amino acid uptake by Saccharomyces cerevisiae, finding that there is a positive control of the general amino acid carrier by a product of the NPR1 gene: the carrier being lost in npr1 mutants140, 375. Also, in 1983, a recessive mutant, per1, was found to prevent inactivation of this carrier by ammonia, although not by glutamine or glutamate76. The measurement of amino acid transport into a number of mutants of Saccharomyces cerevisiae, the mutants in many different combinations (Table 7) enabled Grenson and her colleagues to elucidate some of the highly complex genetic regulatory mechanisms. For example, certain findings gave evidence of two regulatory mechanisms: (a) wild-type cells had no activity of the general amino acid carrier (GAP) when grown in the presence of ammonia, but high activity when grown on proline; (b) on adding ammonia to proline-grown yeast, GAP became progressively less active; (c) this repression of GAP could be reversed for some hours; (d) by mutation, it was possible to lose both these two phenomena, the repression and its reversibility, separately139. Other findings, summarized in Table 7, included the following: In mep1 mutants, ammonia uptake is impaired, so that GAP is active in the presence of NH4+ ions101. In certain mutants of GDH1 (gdhA−), the sensitivity of GAP to ammonia repression decreases146; while gdhCR mutants derepress several ammonia-repressible carriers139. GAP is inactive in the double mutant, gdhA,gdhCR310, because glutamine accumulates in gdhCR mutants in which glutamine synthetase is active218 and this is why GAP is inhibited139. Boris Magasanik has discussed hypotheses about the molecular control underlying the regulation of GAP234. In the late 1960s and 1970s, studies of transport of substances into yeast vacuoles (the largest membrane-bound intracellular structures) were made practicable by the development of techniques for isolating the vacuoles by lysing sphaeroplasts102, 169, 401, 402, as had already been described for the cells of higher plants in 196072. The yeast sphaeroplasts could be lysed by osmotic shock169, 401, mechanically402 or by certain polymers, such as DEAE-dextran (diethylaminoethyl dextran)50 or cytochrome c403. At this time, during the 1960s, it was becoming clear that organelles enable cells to separate different metabolic activities, thus allowing metabolic compartmentation. (a) Cytological observations demonstrated the accumulation in the vacuoles of Candida utilis of S-adenosylmethionine358, 359 (see Figure 23 of article 4, part II, in this series33) and also various other purines16 314, 357. (b) Furthermore, finding certain hydrolases, ribonuclease and peptidases in vacuoles of Saccharomyces cerevisiae, was indicative of their rôle as lysosomes (organelles in which macromolecules are degraded)243. (c) Having found amino acids to be located and stored within the vacuoles of Candida utilis403 and Saccharomyces cerevisiae402, in 1975 Andres Wiemken and his colleagues at Basel characterized, for S. cerevisiae, a specific carrier that took arginine into the vacuoles50. In order to study this carrier, it was necessary to separate the vacuoles; for this, sphaeroplasts were broken up mechanically and the vacuoles isolated by centrifugation in isotonic density gradients (Figure 8). The naked, fragile vacuoles burst on filtering, so for uptake experiments, incubation with labelled arginine was stopped by centrifugation. L-Arginine transport into the vacuoles had saturation kinetics, with a Km of 30 µ M, and was competitively inhibited by D-arginine, L-histidine or L-canavanine50. Wiemken's procedure for separating vacuoles by flotation in isotonic density gradients at 0–4 °C in 10 mM citric acid adjusted to pH 6·8: (A) with 0·6 M D-glucitol; (B) with 0·6 M sucrose. Centrifugation was for 50 min at 5000 × g. ●, intact sphaeroplasts; ○, vacuoles; ·, lipid granules; hatching, soluble fraction402. (Wiemken A, Dürr M. 1974. Characterisation of amino acid pools in the vacuolar compartment of Saccharomyces cerevisiae. Archives of Microbiology 101: 47; Fig. 1. © Springer-Verlag.) This development of methods for measuring uptake by vacuoles made further studies of vacuolar uptake systems practicable, and such studies with Saccharomyces cerevisiae, in the 1970s and 1980s, included those on the uptake of S-adenosyl-L-methionine326, of a proton-carrier Mg2+-ATPase184, 221, 276 and seven independent H+-amino acid antiport carriers, all energized by hydrolysis of ATP322 (Table 9). Ramón Serrano has reviewed some more recent work on the vacuolar ATPases of Saccharomyces cerevisiae332. As indicated above, most of the research on metabolite transport into yeasts has been done with Saccharomyces cerevisiae. However, there has also been a good deal of work on transport into several other species, especially during the 1970s and 1980s. Table 8 lists chronologically, for each of these species, some of this research, particularly that on Candida utilis17, Kluyveromyces lactis18, Rhodotorula glutinis, and Schizosaccharomyces pombe; and a few of the findings are outlined below. Like S. cerevisiae, transport of glucose into K. lactis has been shown to be by facilitated diffusion317, 318: the gene HGT119 encodes a constitutive high-affinity carrier41 with Km = 1 m M394 and RAG120 encodes a low-affinity glucose carrier which is induced by glucose127, Km = 20–50 m M394. Anja Diezemann and Eckhard Boles speculate that Hgt1p may be proton-coupled, as is the fructose carrier Frt1p, which they described [95, p. 287]. K. lactis transports galactose by Lac12p, the sole lactose carrier, lac12 mutants growing on neither sugar305. On the other hand, unlike S. cerevisiae, many species have been shown to take up glucose by active transport. For Candida utilis at low glucose concentrations, glucose enters by proton symport, but it enters by facilitated diffusion at high concentrations287, 378. Various Pichia species were also recorded as having both low- and high-affinity uptake systems for glucose34, 98. In the 1960s and 1970s, Kotyk and Höfer studied a non-fermenting yeast, Rhodosporidium toruloides ( = Rhodotorula sp.), finding that it transports D-glucose actively under aerobic conditions but does not take it up anaerobically202. This work stimulated others to investigate the non-utilization of D-galactose and some glycosides when certain yeasts are anaerobic (the 'Kluyver effect'—see history article no. 927). Indeed, Kluyveromyces thermotolerans ('Torulopsis dattila') was found to require the presence of oxygen for the uptake of D-galactose335. Over several years, Höfer and his colleagues reported on the uptake of various monosaccharides by Schizosaccharomyces pombe, finding them to enter the cells by proton symport160, there being a number of glucose and fructose carriers, Ght1p to Ght6p, Ght3p transporting D-gluconate too157, 222, and there may well be as many hexose carriers to be characterized in this yeast406 as there appear to be in Candida albicans, from the results of screening genomic sequences, reverse-transcription PCR assays, and phylogenetic analyses. The published account of this screening concludes that there are 20 glucose carriers in C. albicans, designating their encoding genes HGT1 to HGT20113. In the 1960s, Gertrudis de la Fuente and Alberto Sols showed that several yeasts, such as S. cerevisiae, hydrolyse certain glycosides outside the plasma membrane, so that it is the component monosaccharides that are transported into the cells91. This is true for many yeasts, the most celebrated examples being sucrose and other β-D-fructofuranosides, as well as melibiose and other α-D-galactopyranosides (for review, see17). However, some other yeasts are known to transport such glycosides into the cytosol, where they are hydrolysed: a mutant strain of S. cerevisiae which lacked external invertase could transport sucrose, so that it was hydrolysed internally by an α-glucosidase187. Now Candida albicans has provided an interesting example of a wild-type yeast which necessarily transports sucrose before hydrolysing it. (a) Some strains of this yeast (and of other species too16, p.186) can utilize sucrose but none use the trisaccharide, raffinose (Figure 9)30p. 84. (b) S. cerevisiae and many other yeasts hydrolyse both raffinose and sucrose outside the plasma membrane by the same β-fructofuranosidase (for review, see [17, pp. 375–378]. (c) The α-glucosidase activity of yeasts is usually cytosolic (for review, see [17, pp. 384–388]. Hence, it seemed probable that C. albicans hydrolyses sucrose by a cytosolic α-glucosidase16, p.186. β-Fructosidase, but not α-glucosidase, hydrolyses the trisaccharide raffinose, liberating fructose; sucrose is a double glycoside (being both β-fructoside and α-glucoside) and hence may be hydrolysed by a β-fructosidase or an α-glucosidase. Figure reproduced from15 Indeed, having shown that C. albicans hydrolyses sucrose cytosolically by an α-glucosidase122, Peter Williamson and his colleagues found sucrose to be transported into the cells by H+ symport404. On the other hand, Schizosaccharomyces pombe can use raffinose [31, p. 678] (by an external invertase?), but appears to have a functional α-glucoside proton symport carrier capable of transporting maltose or sucrose302. Thus, proton symports seem to be usual: for Candida utilis, both glucose and maltose carriers have been shown to involve H+ symport378 and Lac12p, an inducible active transport carrier in Kluyveromyces lactis, takes up lactose93, 351. As long ago as 1910, Candida vini ('Mycoderma cerevisiae') and Pichia membranaefaciens ('Mycoderma valida') were found able to use several carboxylic acids, such as lactic and succinic acids, for growth217. However, although Saccharomyces cerevisiae secretes succinic acid in large amounts during ethanolic fermentation, as Louis Pasteur described in 1860284, oddly enough, this yeast appears to lack carriers which can take up any of the intermediates of the tricarboxylic acid cycle28. Acetate is another matter, Heinrich Wieland and Robert Sonderhoff describing its oxidation by intact cells of S. cerevisiae in 1932400. More recently, in the 1980s and 1990s, an inducible proton symport carrier of S. cerevisiae has been found to mediate the uptake of both D- and L-lactate, pyruvate, propionate, as well as acetate; another carrier taking up formate61, 67. The lactate carrier is encoded by JEN12, 63, 224, 238. Table 8 shows that, especially, in the 1990s, there was research on the uptake of carboxylic acids by at least eight other species and, mostly, this uptake was found to involve active transport by proton symport. The occurrence of active transport of solutes into cells—the energy supplied by metabolism—was established in the 1930s by work on plant roots. At about the same time, various workers also observed the concentration of solutes by animal cells. However, at that time, biochemists generally failed to think of transport as a metabolic activity, as exemplified by the famous cry in 1939 by one of the founders of chemical microbiology, Marjorie Stephenson: 'Don't talk to me about permeability—that is the last resort of the biochemist who cannot find any better explanation'120p. 3. But it was in the 1950s that Jacques Monod and his colleagues laid the foundations of membrane transport as an important part of metabolic studies. After that time, research on transport into yeasts took off, with attempts to find how many carriers there were, as well as their biochemical characteristics, including their specificities and affinities for substrates. The understanding of the nature of active transport depended, of course, on Peter Mitchell's 1961 chemiosmotic hypothesis253 and also Jens Skou's discovery, in 1963, of the rôle of an ATPase in the transfer of ions across cell membranes339. By the 1990s, attention was being given to certain problems of measuring transport kinetics, because of their non-linearity. Using the excellent device shown in Figure 10, devised in Arnošt Kleinzeller's Prague laboratory in the early 1960s, even working with a colleague, the present author could not take samples faster than every 30 s (e.g.36). However, in 1974, Ramón Serrano and Gertrudis DelaFuente took much smaller samples and so were able to bring down the time of sampling to 5 s; they added labelled substrate to 100 µl cell suspension and then stopped the reaction with 10 ml ice-cold water333. This 5 s was considered to be the minimum time feasible for sampling. Stainless steel apparatus for separating cells by membrane filtration, devised in Kleinzeller's laboratory in Prague in the early 1960s. (A) Section through apparatus: heavy cover (1) holds down membrane filter (2) on perforated disc (3). The device was held in a Buchner (side-arm) flask by an air-tight rubber sleeve (4). (B) Photograph of the equipment. Samples of 1 ml yeast suspension (having been incubated with 14C-substrate) were sucked quickly through the filter and washed with two lots of ice-cold 0·1 MKH2PO4. Filter and yeast were then placed in a vial containing scintillation liquid and counted However, commenting on the estimates of Km, despite such improved technique, Günter Fuhrmann and Bernhard Völker (whose analyses are discussed in an earlier article in this series [106, pp. 1050–1052]) wrote: It has become common practice to analyse the sugar transport kinetics from initial uptake rates in Saccharomyces cerevisiae cells with Eadie–Hofstee plots.21 These plots often demonstrate a nonlinear behaviour. They have been resolved incorrectly into two quasilinear components with Km values differing by a factor of about 10. This graphical analysis neglects the obvious additivity of the two hypothetical systems and is therefore in error. A more efficient way to determine kinetic parameters from initial uptake experiments is to use computer-assisted nonlinear regression analysis118p. 180. Consistent with many earlier observations, Bisson and Fraenkel had concluded: … that glucose and fructose uptake in wild-type Saccharomyces may involve two different types of system, distinguishable on the basis of apparent affinity for the sugar … low and high Km systems43. The so-called low affinity system … advocated by Bisson and Fraenkel does probably not exist in reality and has resulted from an erroneous graphical analysis … [118, p. 181]. Nonetheless, since early in the 1980s, most recent developments in understanding metabolite transport have come from applying methods of molecular genetics, rather than from kinetic analyses. Such methods have given evidence of more than 300 membrane transport proteins in Saccharomyces cerevisiae, including those of the mitochondria and vacuoles285, 372. Hexose carriers provide a striking example of these advances. The development of the one-step gene replacement method made it practicable to delete all the HXT genes successively in one and the same strain of S. cerevisiae398 and, thereby, to characterize all the hexose transport proteins of that yeast. Advances of this kind are vital for the detailed understanding of the way transport processes are regulated and integrated with the metabolism of the cells they serve. However, from reading many recent highly sophisticated publications on this subject, it has occurred to the present author that it may sometimes be desirable to remind molecular geneticists that it is the rôle of transport mechanisms in cell physiology that is the raison d'être for their studies! Warmest thanks to Bruno André and A. A. Eddy for their generous help; but they are in no way responsible for the article's faults. As always, the writer is immensely indebted to L. K. Barnett for all the work she has put into improving both writing and illustrations. The Royal Society is also thanked for a research grant.
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