Proton Symport Research Articles

Purification of the Chlorella HUP1 hexose-proton symporter to homogeneity and its reconstitution in vitro.

A prokaryotic biotin acceptor domain was fused to the carboxy terminal end of the Chlorella hexose-proton symporter. The plant symporter is biotinylated in vivo when expressed in Schizosaccharomyces pombe. The extended biotinylated transport protein is fully active, catalyzes accumulation of D-glucose analogs and restores growth of a glucose-uptake-deficient yeast strain. Crude membranes were solubilized with octyl-beta-D-glucoside in the presence of Escherichia coli L-alpha-phosphatidylethanolamine. Biotinylated symporter was purified to homogeneity by biotinavidin affinity chromatography. The symporter protein was reconstituted together with cytochrome-c oxidase prepared from beef heart mitochondria into proteo-liposomes. Cytochrome-c oxidase is a redox-driven H(+)-pump generating a proton motive force (inside negative and alkaline) while transferring electrons from cytochrome-c to oxygen; this energy is used by the symporter to accumulate D-glucose at least 30-fold. In the absence of the driving force the transport protein facilitates diffusion of D-glucose until the concentration equilibrium is reached. It was shown that maximal transport activity depends highly on the amount of co-reconstituted cytochrome-c oxidase and that the symporter possesses 10% of its in vivo turnover number under optimized in vitro transport conditions.

Reconstitution of lactate proton symport activity in plasma membrane vesicles from the yeastCandida utilis

Lactic acid transport was studied in plasma membrane vesicles from the yeast Candida utilis IGC 3092 which were fused with liposomes containing cytochrome c oxidase. After the addition of an electron donor system, these hybrid membrane vesicles were able to generate a proton-motive force of about −150mV, inside alkaline and negative. In vesicles prepared from lactic acid-grown cells, the uptake of labelled lactic acid, at pH 6·2, under energized conditions, was expressed by a kinetics consistent with the involvement of a mediated transport system. This carrier exhibited a substrate specificity pattern identical to the one found for the lactate-proton symport in intact cells. The transport of labelled lactic acid was accumulative and strongly sensitive to the effects of the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, consistent with the involvement of the proton-motive force in acid uptake, hence with the presence of a proton symport for lactate. Dissipation of the transmembrane electric potential by valinomycin did not have a significant effect on lactate accumulation, whereas abolishing the transmembrane pH gradient (ΔpH) by nigericin prevented the accumulation and led to a rapid efflux of the accumulated acid. The data support that the ΔpH is the main component of the proton-motive force involved in the transport of the acid and its accumulation. The lactate-proton symport stoichiometry was 1:1, being independent of the pH. Vesicles prepared from glucose-grown cells did not display the capacity to transport and accumulate lactate. However, activity for the carrier was also reconstituted in vesicles obtained from glucose-grown cells after incubation in buffer containing lactic acid. These results were consistent with those obtained in intact cells, which demonstrated that the lactate-proton symport of the yeast C. utilis is inducible.

Reconstitution of lactate proton symport activity in plasma membrane vesicles from the yeast Candida utilis.

Lactic acid transport was studied in plasma membrane vesicles from the yeast Candida utilis IGC 3092 which were fused with liposomes containing cytochrome c oxidase. After the addition of an electron donor system, these hybrid membrane vesicles were able to generate a proton-motive force of about--150 mV, inside alkaline and negative. In vesicles prepared from lactic acid-grown cells, the uptake of labelled lactic acid, at pH 6.2, under energized conditions, was expressed by a kinetics consistent with the involvement of a mediated transport system. This carrier exhibited a substrate specificity pattern identical to the one found for the lactate-proton symport in intact cells. The transport of labelled lactic acid was accumulative and strongly sensitive to the effects of the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, consistent with the involvement of the proton-motive force in acid uptake, hence with the presence of a proton symport for lactate. Dissipation of the transmembrane electric potential by valinomycin did not have a significant effect on lactate accumulation, whereas abolishing the transmembrane pH gradient (delta pH) by nigericin prevented the accumulation and led to a rapid efflux of the accumulated acid. The data support that the delta pH is the main component of the proton-motive force involved in the transport of the acid and its accumulation. The lactate-proton symport stoichiometry was 1:1, being independent of the pH. Vesicles prepared from glucose-grown cells did not display the capacity to transport and accumulate lactate. However, activity for the carrier was also reconstituted in vesicles obtained from glucose-grown cells after incubation in buffer containing lactic acid. These results were consistent with those obtained in intact cells, which demonstrated that the lactate-proton symport of the yeast C. utilis is inducible.

Blue light‐induced acidification of the medium of a nitrate‐starved Chlorella mutant

Irradiation with blue light resulted in acidification of the suspension medium of a colorless mutant of Chlorella kessleri (Fott et Novákóva, no. 9.80). Qualitatively red light gave the same result, although to a much lower extent than blue, whereas far‐red had little effect. A pulse of blue light led to a transient acidification of the medium. The light‐induced acidification was particularly pronounced if nitrate‐starved cells were used. Acidification was inhibited by vanadate (V2O5, Na3VO4), so that the phenomenon appears due to proton extrusion from the mutant. The light‐induced acidification was also observed in a yellow mutant and the wild‐type of Chlorella kessleri. The role of light in the acidification of the medium of algal cells is discussed in relation to the earlier findings that blue light enhances the uptake of nitrate in the colorless mutant; and that in Chlorella cells the transport process of nitrate is connected with a proton symport.

Characterization of Glutathione Uptake in Broad Bean Leaf Protoplasts.

Transport of reduced glutathione (GSH) and oxidized glutathione (GSSG) was studied with broad bean (Vicia faba L.) leaf tissues and protoplasts. Protoplasts and leaf discs took up GSSG at a rate about twice the uptake rate of GSH. Detailed studies with protoplasts indicated that GSH and GSSG uptake exhibited the same sensitivity to the external pH and to various chemical reagents. GSH uptake was inhibited by GSSG and glutathione conjugates. GSSG uptake was inhibited by GSH and GS conjugates, and the uptake of metolachlor-GS was inhibited by GSSG. Various amino acids (L-glutamic acid, L-glutamine, L-cysteine, L-glycine, L-methionine) and peptides (glycine-glycine, glycine-glycine-glycine) affected neither the transport of GSH nor GSSG. Uptake kinetics indicate that GSH is taken up by a single saturable transporter, with an apparent Km of 0.4 mM, whereas GSSG uptake exhibits two saturable phases, with an apparent Km of 7 [mu]M and 3.7 mM. It is concluded that the plasma membrane of leaf cells contains a specific transport system for glutathione, which takes up GSSG and GS conjugates preferentially over GSH. Proton flux measurements and electrophysiological measurements indicate that GSH and GSSG are taken up with proton symport. However, a detailed analysis of these measurements suggests that the ion movements induced by GSSG differ from those induced by GSH.

Kinetics, energetics and specificity of a general amino acid transporter from the ectomycorrhizal fungus Paxillus involutus

The kinetics, energetics and specificity of a general amino acid transporter were studied in the ectomycorrhizal fungus Paxillus involutus (Batsch) Fr. The uptake of amino acids showed features characteristic of active transport. After correction for a non-mediated transport component, the kinetics of glutamate, glutamine, alanine and aspartate uptake measured over a wide concentration range followed the simple Michaelis-Menten saturation curves. The apparent K m derived from the Eadie-Hofstee plots ranged from 7 μM for alanine to 27 μM for glutamate. Dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone and NaN3 strongly inhibited amino acid uptake, whereas dicyclohexylcarbodiimide, vanadate and the ionophores monensin and nonactin had no effect on the uptake. Both pH dependence and inhibition by protonophores are consistent with a proton symport mechanism for amino acid uptake by P. involutus. Competition studies indicated a broad substrate recognition by the uptake system, which resembles the general amino acid permease of yeast. Dixon plots of the inhibition of glutamate uptake by alanine, lysine and methionine sulfoximine showed that inhibitions were competitive. The physiological importance of this transporter for the exchange of nitrogenous compounds between fungal and host plant cells in ectomycorrhizal associations is discussed.

Characterization of the lactose transport system in the strain Bifidobacterium bifidum DSM 20082.

Lactose was fermented but not assimilated by the strain Bifidobacterium bifidum DSM 20082. The sugar uptake was measured with lactose 14C. Km and V(max) values were respectively 2.6 mM and 12.11 nmol/min/mg of cell protein. The lactose transport system and the beta-D-galactosidase were stimulated when the cells were grown with lactose, but isopropyl-beta-D-thiogalactopyranoside had no effect. Lactose uptake was inhibited by compounds which interfered with proton and metal ionophore. Na+, Li+, or K+ did not affect incorporation of lactose. Furthermore, the lactose uptake decreased when an inhibitor of ATP synthesis was used. From the results of this study, the stain contained an active lactose transport system, probably a proton symport as described for Escherichia coli but with a different regulation system.

Nickel(II) Complex of 6,6′-Bis(hexylbenzoylamino)-2,2′-bipyridine as a Functional Carrier: pH-Induced Affinity-Switching and Mechanism of the SCN−-Selective Up-Hill Transport

Abstract The Ni(II) complex of 6,6′-bis(hexylbenzoylamino)-2,2′-bipyridine (LH2) in square-planar symmetry underwent a reversible and simultaneous deprotonation of the two amide units. The non-deprotonated complex (NiLH22+) showed high SCN− selectivity by the preferential axial coordination of two SCN− molecules (log KSCN = 8.11 ± 0.03 in CH2Cl2 at 20 °C), while the deprotonated complex showed little affinity toward anions. Deprotonation-induced affinity-switching of the complex by the external aqueous layer mediated an efficient SCN−-selective up-hill transport across a CH2Cl2 liquid membrane by coupling with the symport of protons from pH 4.0 to 6.0 aqueous solutions. Equilibrium at the aqueous/organic interface could be represented by the equation Kapp = ([Ni(LH2)(SCN)2]org)/([NiL]org[H+]2aq[SCN−]2aq), where Kapp is the equilibrium constant (log Kapp = 13.6 at 20 °C). However, the rates of protonation and deprotonation of the complex at the interface were dependent mostly on the concentration of the complex in the organic layer. Based on these results, detailed mechanisms of the transport are discussed.

Sugar uptake by the dermal transfer cells of developing cotyledons of Vicia faba L. : Mechanism of energy coupling.

The mechanism of carrier-mediated sucrose uptake by the dermal transfer cells of developing Vicia faba L. cotyledons was studied using excised cotyledons and isolated transfer cell protoplasts. Addition of sucrose resulted in a transitory alkalinization of the bathing solution whereas additions of glucose, fructose or raffinose had no effect. Dissipating the proton motive force by exposing cotyledons and isolated transfer cell protoplasts to an alkaline pH, carbonylcyanide m-chlorophenylhydrazone, weak acids (propionic acid and 5,5'-dimethyl-oxazolidine-2,4-dione) or tetraphenylphos-phonium ion resulted in a significant reduction of sucrose uptake. The ATPase inhibitors, erythrosin B (EB), diethylstilbestrol (DES) and N,N'-dicyclohexylcarbodiimide (DCCD) were found to abolish the sucrose-induced medium alkanization as well as reduce sucrose uptake. Cytochemical localization of the ATPase, based on lead precipitation, demonstrated that the highest activity was present in the plasma membranes located in wall ingrowth regions of the dermal transfer cells. The presence of a transplasma-membrane redox system was detected by the extracellular reduction of the electron acceptor, hexacyanoferrate III. The reduction of the ferric ion was coupled to a release of protons. The redox-induced proton extrusion was abolished by the ATPase inhibitors EB, DES and DCCD suggesting that proton extrusion was solely through the H+-ATPase. Based on these findings, it is postulated that cotyledonary dermal transfer cells take up sucrose by a proton symport mechanism with the proton motive force being generated by a H + -ATPase. Sucrose uptake by the storage parenchyma and inner epidermal cells of the cotyledons did not exhibit characteristics consistent with sucrose-proton symport.

Validation of a metabolic network for Saccharomyces cerevisiae using mixed substrate studies.

Setting up a metabolic network model for respiratory growth of Saccharomyces cerevisiae requires the estimation of only two (energetic) stoichiometric parameters: (1) the operational PO ratio and (2) a growth-related maintenance factor k. It is shown, both theoretically and practically, how chemostat cultivations with different mixtures of two substrates allow unique values to be given to these unknowns of the proposed metabolic model. For the yeast and model considered, an effective PO ratio of 1.09 mol of ATP/mol of O (95% confidence interval 1.07-1.11) and a k factor of 0.415 mol of ATP/C-mol of biomass (0.385-0.445) were obtained from biomass substrate yield data on glucose/ethanol mixtures. Symbolic manipulation software proved very valuable in this study as it supported the proof of theoretical identifiability and significantly reduced the necessary computations for parameter estimation. In the transition from 100% glucose to 100% ethanol in the feed, four metabolic regimes occur. Switching between these regimes is determined by cessation of an irreversible reaction and initiation of an alternative reaction. Metabolic network predictions of these metabolic switches compared well with activity measurements of key enzymes. As a second validation of the network, the biomass yield of S. cerevisiae on acetate was also compared to the network prediction. An excellent agreement was found for a network in which acetate transport was modeled with a proton symport, while passive diffusion of acetate gave significantly higher yield predictions.

Ecology, Metabolism, and Genetics of Ruminal Selenomonads

Selenomonas ruminantium is one of the more prominent and functionally diverse bacteria present in the rumen and can survive under a wide range of nutritional fluctuations. Selenomonas is not a degrader of complex polysaccharides associated with dietary plant cell wall components, but is important in the utilization of soluble carbohydrates released from initial hydrolysis of these polymers by other ruminal bacteria. Selenomonads have multiple carbon flow routes for carbohydrate catabolism and ATP generation, and subspecies differ in their ability to use lactate. Some soluble carbohydrates (glucose, sucrose) appear to be transported via the phosphoenolpyruvate phosphotransferase system, while arabinose and xylose are transported by proton symport. High cell yields and the presence of electron transport components in Selenomonas strains has been documented repeatedly and this may partially account for the energy partitioning observed between energy consumed for growth and maintenance functions. Most strains can utilize ammonia, protein, and/or amino acids as a nitrogen source. Some strains can hydrolyze urea and/or reduce nitrate and use the ammonia for the biosynthesis of amino acids. Experimental evidence suggests that ammonia assimilatory enzymes in some strains may possess unique properties with respect to other presumably similar bacteria. Little is known about the genetics of ruminal selenomonads. Plasmid DNA has been isolated from some strains, but it is unknown what physiological functions may be encoded on these extrachromosomal elements. Due to the predominance of S. ruminantium in the rumen, it is an ideal candidate for genetic manipulation. Once the genetics of this bacterium are better understood, it may be possible to amplify its role in the rumen.

Utilization of short-chain monocarboxylic acids by the yeast Torulaspora delbrueckii: Specificity of the transport systems and their regulation

Cells of Torulaspora delbrueckii IGC 4478 grown in a medium with DL-lactic acid (0.5%, v/v, at pH 5.0) exhibited Michaelis-Menten kinetics for labelled l-lactic acid transport with the following parameters at pH 5.0: V max, 0.38 nmol of total l-lactic acid s −1 per mg dry weight of cells and K m, 0.05 mM total l-lactic acid. Furthermore, evidence was available indicating that a proton symport for the charged form of the acid was involved. d-lactic, acetic, propionic, pyruvic and formic acids were competitive inhibitors of labelled l-lactic acid transport, suggesting that these acids used the same transport system. The ability of T. delbrueckii IGC 4478 to grow with acetic acid as the carbon source was dependent on the acid concentration and on the pH of the culture medium. When the cells were grown in 0.5% (v/v) acetic acid (pH 6.0), the transport of labelled acetic acid followed a Michaelis-Menten kinetics with the following parameters at pH 5.0: V max , 2.93 nmol of total acetic acid s −1 1 per mg dry weight of cells and K m, 0.55 mM total acetic acid. The system a displayed a behavior consistent with a proton symport mechanism. However, the specificity of this carrier was distinct from that observed for the monocarboxylate transport in dl-lactic acid grown cells. While propionic and formic acids were competitive inhibitors of the labelled acetic acid transport, dl-lactic and pyruvic acids did not exhibit any inhibitory effects on that transport. Moreover, under the same conditions, no uptake was observed when the transport was measured with labelled l-lactic acid. Both systems were inducible and subjected to repression by glucose, fructose or sucrose. Accordingly, diauxic growth was observed in a medium containing a mixture of any of these sugars plus lactic, pyruvic or acetic acid. While the induction of the acetate proton-symport appeared to be exclusively associated with acetic acid, the lactate proton-symport could be induced by either lactic or pyruvic acid but not by acetic acid. Besides, glucose repressed cells were still permeable to the undissociated form of the acids which entered the cells by simple diffusion. Furthermore, the activities of the lactate proton-symport and of the acetate proton-symport appeared not to be associated with the activity of the l-lactate (cytochrome) dehydrogenase.

Kinetic Analysis of Lactose and Proton Coupling in Glu379 Mutants of the Lactose Transport Protein of Streptococcus thermophilus

The role of Glu379 in the lactose-H+ symport protein (LacS) of Streptococcus thermophilus was studied by analyzing the kinetic mechanism of transport of wild-type and Ala379, Asp379, and Gln379 mutant proteins. Glu379 forms part of the sequence motif Lys-X-X-His-X-X-Glu that is present in a number of sugar transport proteins, including LacY of Escherichia coli. The E379A and E379Q mutants were defective in the uptake of lactose against a concentration gradient and lactose-dependent proton uptake, but catalyzed facilitated influx of lactose down a concentration gradient and equilibrium exchange with rates similar to that of the wild-type enzyme. The E379D mutant was partially defective in the coupled transport of lactose and protons. These results suggest that an acidic residue at position 379 is required for the coupled uptake of lactose and protons and are consistent with a mechanism in which lactose transport in the E379A and E379Q mutants occurs by uniport rather than proton symport. Lactose efflux down a concentration gradient in wild-type LacS and LacS-E379D increased with pH with apparent pK (pKa) values of > or = 8.5 and 8.0, respectively, whereas efflux in the E379Q mutant increased sigmoidally with a pKa of about 6.0. Imposition of an artificial membrane potential (inside negative) in membrane vesicles bearing wild-type LacS or LacS-E379Q not only inhibited the lactose efflux mediated by wild-type but also that of the mutant enzyme. To associate the role of Glu379 with specific step(s) in the translocation cycle of LacS, the properties of wild-type LacS and the Glu379 mutants have been evaluated by numerical analysis of simple kinetic schemes for translocation catalysis by solute H+ symport proteins. The properties of the wild-type enzyme are consistent with a mechanism in which the order of ligand binding on the inside is substrate first and proton last, whereas the order is random (or proton first, substrate last) at the outer surface of the membrane. The wild-type enzyme is asymmetric with regard to proton binding; the pK for proton binding on the outside is at least 4 units higher than the pK on the inside. The properties of the Glu379 mutants correspond with a lowering of the pK on the outside (pKOUT approximately pKIN), and the induction of a leak pathway in which the binary enzyme-substrate complex becomes mobile.

The plasma membrane of Saccharomyces cerevisiae: structure, function, and biogenesis

The composition of phospholipids, sphingolipids, and sterols in the plasma membrane has a strong influence on the activity of the proteins associated or embedded in the lipid bilayer. Since most lipid-synthesizing enzymes in Saccharomyces cerevisiae are located in intracellular organelles, an extensive flux of lipids from these organelles to the plasma membrane is required. Although the pathway of protein traffic to the plasma membrane is similar to that of most of the lipids, the bulk flow of lipids is separate from vesicle-mediated protein transport. Recent advances in the analysis of membrane budding and membrane fusion indicate that the mechanisms of protein transport from the endoplasmic reticulum to the Golgi and from the Golgi to plasma membrane are similar. The majority of plasma membrane proteins transport solutes across the membrane. A number of ATP-dependent export systems have been detected that couple the hydrolysis of ATP to transport of molecules out of the cell. The hydrolysis of ATP by the plasma membrane H(+)-ATPase generates a proton motive force which is used to drive secondary transport processes. In S. cerevisiae, many substrates are transported by more than one system. Transport of monosaccharide is catalyzed by uniport systems, while transport of disaccharides, amino acids, and nucleosides is mediated by proton symport systems. Transport activity can be regulated at the level of transcription, e.g., induction and (catabolite) repression, but transport proteins can also be affected posttranslationally by a process termed catabolite inactivation. Catabolite inactivation is triggered by the addition of fermentable sugars, intracellular acidification, stress conditions, and/or nitrogen starvation. Phosphorylation and/or ubiquitination of the transport proteins has been proposed as an initial step in the controlled inactivation and degradation of the target enzyme. The use of artificial membranes, like secretory vesicles and plasma membranes fused with proteoliposomes, as model systems for studies on the mechanism and regulation of transport is evaluated.

Lack of lactate-proton symport activity in pck1 mutants of Saccharomyces cerevisiae

Mutants of Saccharomyces cerevisiae without phosphoenolpyruvate carboxykinase activity showed no measurable lactate proton symport, while mutants without fructose-1,6-bisphosphatase had normal transport activity. Incubation of a pck1 mutant, under derepression conditions in the presence of glycerol, restored the activity of the lactate-proton symport, with identical kinetic characteristics to that in the wild-type. For efficient lactate-proton symport activity, not only is an external inducer such as lactic acid needed, but also a molecule derived from the acid metabolism may be necessary.

Lack of lactate-proton symport activity in pck1 mutants of Saccharomyces cerevisiae.

Mutants of Saccharomyces cerevisiae without phosphoenolpyruvate carboxykinase activity showed no measurable lactate proton symport, while mutants without fructose-1,6-bisphosphatase had normal transport activity. Incubation of a pck1 mutant, under derepression conditions in the presence of glycerol, restored the activity of the lactate-proton symport, with identical kinetic characteristics to that in the wild-type. For efficient lactate-proton symport activity, not only is an external inducer such as lactic acid needed, but also a molecule derived from the acid metabolism may be necessary.

Allosteric regulation of the glucose:H+ symporter of Lactobacillus brevis: cooperative binding of glucose and HPr(ser-P)

Lactobacillus brevis transports glucose and the nonmetabolizable glucose analog 2-deoxyglucose via a proton symport mechanism that is allosterically inhibited by the seryl-phosphorylated derivative of HPr, the small phosphocarrier protein of the phosphotransferase system. We have demonstrate that S46DHPr, a mutant analog of HPr which conformationally resembles HPr(ser-P) but not free HPr, specifically binds to membranes derived from glucose-grown L. brevis cells if and only if a substrate of the glucose permease is also present.

Characterization of amino-acid transport in Ricinus communis roots using isolated membrane vesicles

The mechanism and specificity of amino-acid transport at the plasma membrane of Ricinus communis L. roots was investigated using membrane vesicles isolated by phase partitioning. The transport of glutamine, isoleucine, glutamic acid and aspartic acid was driven by both a pH gradient and a membrane potential (internally alkaline and negative), created artificially across the plasma membrane. This is consistent with transport via a proton symport. In contrast, the transport of the basic amino acids, lysine and arginine, was driven by a negative internal membrane potential but not by a pH gradient, suggesting that these amino acids may be taken up via a voltage-driven uniport. The energized uptake of all of the amino acids tested showed a saturable phase, consistent with carrier-mediated transport. In addition, the membrane-potential-driven transport of all the amino acids was greater at pH 5.5 than at pH 7.5, which suggests that there could be a direct pH effect on the carrier. Several amino-acid carriers could be resolved, based on competition studies: a carrier with a high affinity for a range of neutral amino acids (apart from asparagine) but with a low affinity for basic and acidic amino acids; a carrier which has a high affinity for a range of neutral amino acids except isoleucine and valine, but with a low affinity for basic and acidic amino acids; and a carrier which has a higher affinity for basic and some neutral amino acids but has a lower affinity for acidic amino acids. The existence of a separate carrier for acidic amino acids is discussed.

The Significance of Active Fructose Transport and Maximum Temperature for Growth in the Taxonomy of Saccharomyces sensu stricto

Summary Eighty five strains representing the four sibling species currently recognized within the Saccharomyces sensu stricto group were tested for the capacity to transport fructose by an active proton symport. Active transport of fructose was not detected in the strains of Sacch. cerevisiae and Sacch. paradoxus , whereas it was found in the strains assigned to Sacch. bayanus and Sacch. pastorianus . The maximum temperature for growth (T max ) of the latter two species ranged from 30°C to 34°C and from 28°C to 35°C respectively, at variance with the ranges of T max values of Sacch. cerevisiae (35 δC–43 δC, with one exception showing a lower T max ) and Sacch. paradoxus (35 °C–41 °C). The identity of the strains has been defined by DNA/DNA reassociation studies and their genome size estimated from the kinetics of DNA renaturation. The data support earlier reports of the intraspecific heterogeneity of Sacch. cerevisiae and of close relationships between species. This work represents an extension of our initial investigation of sibling Saccharomyces species. While showing that active fructose transport is a suitable systematic character for rapid differentiation of the two subgroups within Saccharomyces sensu stricto , useful differential tests to be applied within each subgroup were not found.

The plasma membrane of Saccharomyces cerevisiae: structure, function, and biogenesis.

The composition of phospholipids, sphingolipids, and sterols in the plasma membrane has a strong influence on the activity of the proteins associated or embedded in the lipid bilayer. Since most lipid-synthesizing enzymes in Saccharomyces cerevisiae are located in intracellular organelles, an extensive flux of lipids from these organelles to the plasma membrane is required. Although the pathway of protein traffic to the plasma membrane is similar to that of most of the lipids, the bulk flow of lipids is separate from vesicle-mediated protein transport. Recent advances in the analysis of membrane budding and membrane fusion indicate that the mechanisms of protein transport from the endoplasmic reticulum to the Golgi and from the Golgi to plasma membrane are similar. The majority of plasma membrane proteins transport solutes across the membrane. A number of ATP-dependent export systems have been detected that couple the hydrolysis of ATP to transport of molecules out of the cell. The hydrolysis of ATP by the plasma membrane H(+)-ATPase generates a proton motive force which is used to drive secondary transport processes. In S. cerevisiae, many substrates are transported by more than one system. Transport of monosaccharide is catalyzed by uniport systems, while transport of disaccharides, amino acids, and nucleosides is mediated by proton symport systems. Transport activity can be regulated at the level of transcription, e.g., induction and (catabolite) repression, but transport proteins can also be affected posttranslationally by a process termed catabolite inactivation. Catabolite inactivation is triggered by the addition of fermentable sugars, intracellular acidification, stress conditions, and/or nitrogen starvation. Phosphorylation and/or ubiquitination of the transport proteins has been proposed as an initial step in the controlled inactivation and degradation of the target enzyme. The use of artificial membranes, like secretory vesicles and plasma membranes fused with proteoliposomes, as model systems for studies on the mechanism and regulation of transport is evaluated.