Kinetics of active alpha-glucoside transport in Saccharomyces cerevisiae

alpha-Glucosides are the most abundant fermentable sugars in the industrial applications of Saccharomyces cerevisiae, and the active transport across the plasma membrane is the rate-limiting step for their metabolism. In this report we performed a detailed kinetic analysis of the active alpha-glucoside transport system(s) present in a wild-type strain, and in strains with defined alpha-glucoside permeases. Our results indicate that the wild-type strain harbors active transporters with high and low affinity for maltose and trehalose, and low-affinity transport systems for maltotriose and alpha-methylglucoside. The maltose permease encoded by the MAL21 gene showed a high affinity (K(m) approximately 5 mM) for maltose, and a low affinity (K(m) approximately 90 mM) for trehalose. On the other hand, the alpha-glucoside permease encoded by the AGT1 gene had a high affinity (K(m) approximately 7 mM) for trehalose, a low affinity (K(m) approximately 18 mM) for maltose and maltotriose, and a very low affinity (K(m) approximately 35 mM) for alpha-methylglucoside.     

Characterization and functional analysis of the MAL and MPH Loci for maltose utilization in some ale and lager yeast strains

Maltose and maltotriose are the major sugars in brewer's wort. Brewer's yeasts contain multiple genes for maltose transporters. It is not known which of these express functional transporters. We correlated maltose transport kinetics with the genotypes of some ale and lager yeasts. Maltose transport by two ale strains was strongly inhibited by other alpha-glucosides, suggesting the use of broad substrate specificity transporters, such as Agt1p. Maltose transport by three lager strains was weakly inhibited by other alpha-glucosides, suggesting the use of narrow substrate specificity transporters. Hybridization studies showed that all five strains contained complete MAL1, MAL2, MAL3, and MAL4 loci, except for one ale strain, which lacked a MAL2 locus. All five strains also contained both AGT1 (coding a broad specificity alpha-glucoside transporter) and MAL11 alleles. MPH genes (maltose permease homologues) were present in the lager but not in the ale strains. During growth on maltose, the lager strains expressed AGT1 at low levels and MALx1 genes at high levels, whereas the ale strains expressed AGT1 at high levels and MALx1 genes at low levels. MPHx expression was negligible in all strains. The AGT1 sequences from the ale strains encoded full-length (616 amino acid) polypeptides, but those from both sequenced lager strains encoded truncated (394 amino acid) polypeptides that are unlikely to be functional transporters. Thus, despite the apparently similar genotypes of these ale and lager strains revealed by hybridization, maltose is predominantly carried by AGT1-encoded transporters in the ale strains and by MALx1-encoded transporters in the lager strains.     

Sucrose fermentation by Saccharomyces cerevisiae lacking hexose transport

Sucrose is the major carbon source used by Saccharomyces cerevisiae during production of baker's yeast, fuel ethanol and several distilled beverages. It is generally accepted that sucrose fermentation proceeds through extracellular hydrolysis of the sugar, mediated by the periplasmic invertase, producing glucose and fructose that are transported into the cells and metabolized. In the present work we analyzed the contribution to sucrose fermentation of a poorly characterized pathway of sucrose utilization by S. cerevisiae cells, the active transport of the sugar through the plasma membrane and its intracellular hydrolysis. A yeast strain that lacks the major hexose transporters (hxt1-hxt7 and gal2) is incapable of growing on or fermenting glucose or fructose. Our results show that this hxt-null strain is still able to ferment sucrose due to direct uptake of the sugar into the cells. Deletion of the AGT1 gene, which encodes a high-affinity sucrose-H(+) symporter, rendered cells incapable of sucrose fermentation. Since sucrose is not an inducer of the permease, expression of the AGT1 must be constitutive in order to allow growth of the hxt-null strain on sucrose. The molecular characterization of active sucrose transport and fermentation by S. cerevisiae cells opens new opportunities to optimize yeasts for sugarcane-based industrial processes.     

Identification and characterization of the maltose permease in genetically defined Saccharomyces strain

Saccharomyces yeasts ferment several alpha-glucosides including maltose, maltotriose, turanose, alpha-methylglucoside, and melezitose. In the utilization of these sugars transport is the rate-limiting step. Several groups of investigators have described the characteristics of the maltose permease (D. E. Kroon and V. V. Koningsberger, Biochim. Biophys. Acta 204:590-609, 1970; R. Serrano, Eur. J. Biochem. 80:97-102, 1977). However, Saccharomyces contains multiple alpha-glucoside transport systems, and these studies have never been performed on a genetically defined strain shown to have only a single permease gene. In this study we isolated maltose-negative mutants in a MAL6 strain and, using a high-resolution mapping technique, we showed that one class of these mutants, the group A mutants, mapped to the MAL61 gene (a member of the MAL6 gene complex). An insertion into the N-terminal-coding region of MAL61 resulted in the constitutive production of MAL61 mRNA and rendered the maltose permease similarly constitutive. Transformation by high-copy-number plasmids containing the MAL61 gene also led to an increase in the maltose permease. A deletion-disruption of MAL61 completely abolished maltose transport activity. Taken together, these results prove that this strain has only a single maltose permease and that this permease is the product of the MAL61 gene. This permease is able to transport maltose and turanose but cannot transport maltotriose, alpha-methylglucoside, or melezitose. The construction of strains with only a single permease will allow us to identify other maltose-inducible transport systems by simple genetic tests and should lead to the identification and characterization of the multiple genes and gene products involved in alpha-glucoside transport in Saccharomyces yeasts.     

Characterization of Schizosaccharomyces pombe malate permease by expression in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, L-malic acid transport is not carrier mediated and is limited to slow, simple diffusion of the undissociated acid. Expression in S. cerevisiae of the MAE1 gene, encoding Schizosaccharomyces pombe malate permease, markedly increased L-malic acid uptake in this yeast. In this strain, at pH 3.5 (encountered in industrial processes), L-malic acid uptake involves Mae1p-mediated transport of the monoanionic form of the acid (apparent kinetic parameters: Vmax = 8.7 nmol/mg/min; Km = 1.6 mM) and some simple diffusion of the undissociated L-malic acid (Kd = 0.057 min(-1)). As total L-malic acid transport involved only low levels of diffusion, the Mae1p permease was further characterized in the recombinant strain. L-Malic acid transport was reversible and accumulative and depended on both the transmembrane gradient of the monoanionic acid form and the DeltapH component of the proton motive force. Dicarboxylic acids with stearic occupation closely related to L-malic acid, such as maleic, oxaloacetic, malonic, succinic and fumaric acids, inhibited L-malic acid uptake, suggesting that these compounds use the same carrier. We found that increasing external pH directly inhibited malate uptake, resulting in a lower initial rate of uptake and a lower level of substrate accumulation. In S. pombe, proton movements, as shown by internal acidification, accompanied malate uptake, consistent with the proton/dicarboxylate mechanism previously proposed. Surprisingly, no proton fluxes were observed during Mae1p-mediated L-malic acid import in S. cerevisiae, and intracellular pH remained constant. This suggests that, in S. cerevisiae, either there is a proton counterflow or the Mae1p permease functions differently from a proton/dicarboxylate symport.     

A Novel Sinorhizobium meliloti Operon Encodes an α-Glucosidase and a Periplasmic-Binding-Protein-Dependent Transport System for α-Glucosides

The most abundant carbon source transported into legume root nodules is photosynthetically produced sucrose, yet the importance of its metabolism by rhizobia in planta is not yet known. To identify genes involved in sucrose uptake and hydrolysis, we screened a Sinorhizobium meliloti genomic library and discovered a segment of S. meliloti DNA which allows Ralstonia eutropha to grow on the alpha-glucosides sucrose, maltose, and trehalose. Tn5 mutagenesis localized the required genes to a 6.8-kb region containing five open reading frames which were named agl, for alpha-glucoside utilization. Four of these (aglE, aglF, aglG, and aglK) appear to encode a periplasmic-binding-protein-dependent sugar transport system, and one (aglA) appears to encode an alpha-glucosidase with homology to family 13 of glycosyl hydrolases. Cosmid-borne agl genes permit uptake of radiolabeled sucrose into R. eutropha cells. Analysis of the properties of agl mutants suggests that S. meliloti possesses at least one additional alpha-glucosidase as well as a lower-affinity transport system for alpha-glucosides. It is possible that the Fix+ phenotype of agl mutants on alfalfa is due to these additional functions. Loci found by DNA sequencing to be adjacent to aglEFGAK include a probable regulatory gene (aglR), zwf and edd, which encode the first two enzymes of the Entner-Doudoroff pathway, pgl, which shows homology to a gene encoding a putative phosphogluconolactonase, and a novel Rhizobium-specific repeat element.     

Sugar transport by the bacterial phosphotransferase system. Regulation of other transport systems (lactose and melibiose)

The role of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) in the phenomenon of inducer exclusion was examined in whole cells of Salmonella typhimurium which carried the genes of the Escherichia coli lactose operon on an episome. In the presence of the PTS substrate methyl alpha-D-glucopyranoside, the extent of accumulation of the lactose analog methyl beta-D-thiogalactopyranoside was reduced. A strain carrying a mutation in the gene for Enzyme I was hypersensitive to the PTS effect, while a crr mutant strain was completely resistant. Influx, efflux, and exchange of galactosides via the lactose "permease" were inhibited by methyl alpha-glucoside. This inhibition occurred in the presence of metabolic energy poisons, and therefore does not involve either the generation of metabolic energy or energy-coupling to the lactose transport system. When the cellular content of the lactose permease was increased by induction with isopropyl beta-D-thiogalactopyranoside, cells gradually became less sensitive to inducer exclusion. The extent of inhibition of methyl beta-thiogalactoside accumulation by methyl alpha-glucoside was shown to be dependent on the relative cellular content of the PTS and lactose system. The data were consistent with an hypothesis involving partial inactivation of galactoside transport due to interaction between a component of the PTS and the lactose permease. By examination of the effects of the PTS and lactose uptake and melibiose permease-mediated uptake of methyl beta-thiogalactoside, it was further shown that the manner in which inducer exclusion is expressed is independent on the routes available to the non-PTS sugar for exit from the cell.     

AGT1, Encoding an α-Glucoside Transporter Involved in Uptake and Intracellular Accumulation of Trehalose in Saccharomyces cerevisiae

The trehalose content in Saccharomyces cerevisiae can be significantly manipulated by including trehalose at an appropriate level in the growth medium. Its uptake is largely dependent on the expression of AGT1, which encodes an alpha-glucoside transporter. The trehalose found in a tps1 mutant of trehalose synthase may therefore largely reflect its uptake from the enriched medium that was employed.     

The disruption of JEN1 from Candida albicans impairs the transport of lactate

A lactate permease was biochemically identified in Candida albicans RM1000 presenting the following kinetic parameters at pH 5.0: Km 0.33+/-0.09 mM and Vmax 0.85+/-0.06 nmol s(-1) mg dry wt(-1). Lactate uptake was competitively inhibited by pyruvic and propionic acids; acetic acid behaved as a non-competitive substrate. An open reading frame (ORF) homologous to Saccharomyces cerevisiae gene JEN1 was identified (CaJEN1). Deletions of both CaJEN1 alleles of C. albicans (resulting strain CPK2) resulted in the loss of all measurable lactate permease activity. No CaJEN1 mRNA was detectable in glucose-grown cells neither activity for the lactate transporter. In a medium containing lactic acid, CaJEN1 mRNA was detected in the RM1000 strain, and no expression was found in cells of CPK2 strain. In a strain deleted in the CaCAT8 genes the expression of CaJEN1 was significantly reduced, suggesting the role of this gene as an activator for CaJEN1 expression. Both in C. albicans and in S. cerevisiae cells CaJEN1-GFP fusion was expressed and targeted to the plasma membrane. The native CaJEN1 was not functional in a S. cerevisiae jen1delta strain. Changing ser217-CTG codon (encoding leucine in S. cerevisiae) to a TCC codon restored the permease activity in S. cerevisiae, proving that the CaJEN1 gene codes for a monocarboxylate transporter.     

Kinetic properties of yeast lysine permeases coded by genes on multi-copy vectors

Abstract Amplification of the LYP1 transport system in the plasma membrane of Saccharomyces cerevisiae did not change the substrate specificity, the affinity and the pH optimum of the transport system for lysine, but significantly increased the uptake velocity and accumulation ratio. The dependence of active lysine uptake and accumulation on pH is probably given by the properties of the permease itself rather than by the value of available protonmotive force.     

Cloning of Pichia pastoris Fet3: insights into the high affinity iron uptake system

High-affinity iron uptake by yeast cells appears to require the presence of a complex formed on the plasma membrane by the multicopper oxidase Fet3 and the permease Ftr1 which work together to allow iron to enter safely inside the cell. The Pichia pastoris ferroxidase Fet3 has been cloned and it has been found to display high sequence similarity to other yeast multicopper oxidases, including all the predicted ligands for the catalytic copper atoms and for the iron substrate. P. pastoris appears to possess a high-affinity iron uptake system similar to that of S. cerevisiae, as far as regulation of expression is concerned. However, the P. pastoris high-affinity iron uptake system presents a K(m) value for iron almost ten times higher than that of S. cerevisiae, possibly to control iron fluxes over a wider range of concentrations of this metal, in order to avoid toxic iron overloading.     

Improvement of maltotriose fermentation by Saccharomyces cerevisiae

AIMS: To enhance the fermentation of maltotriose by industrial Saccharomyces cerevisiae strains. METHODS AND RESULTS: The capability to ferment maltotriose by an industrial yeast strain that uses this sugar aerobically was tested in shake flasks containing rich medium. While the presence of maltose in the medium did not improve maltotriose fermentation, enhanced and constitutive expression of the AGT1 permease not only increased the uptake of maltotriose, but allowed efficient maltotriose fermentation by this strain. Supplementation of the growth medium with 20 mmol magnesium l(-1) also increased maltotriose fermentation. CONCLUSIONS: Over expression of the AGT1 permease and magnesium supplementation improved maltotriose fermentation by an industrial yeast strain that respired but did not ferment this sugar. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to the elucidation of the roles of the AGT1 permease and nutrients in the fermentation of all sugars present in starch hydrolysates, a highly desirable trait for several industrial yeasts.     

Transport by the lactose permease of Escherichia coli as the basis of lactose killing.

Lactose killing is a peculiar phenomenon in which 80 to 98% of the Escherichia coli cells taken from a lactose-limited chemostat die when plated on standard lactose minimal media. This unique form of suicide is caused by the action of the lactose permease. Since uptake of either lactose or galactose by the lactose permease caused death, the action of rapid transport across the membrane must be the cause of the phenomenon. Alternative causes of lactose killing, such as accumulation of toxic metabolic intermediates or action of the beta-galactosidase, have been eliminated. It is proposed that rapid uptake of sugars by the lactose permease disrupts membrane function, perhaps causing collapse of the membrane potential.     

Properties of yeast Saccharomyces cerevisiae plasma membrane dicarboxylate transporter

Transport of succinate into Saccharomyces cerevisiae cells was determined using the endogenous coupled mitochondrial succinate oxidase system. The dependence of succinate oxidation rate on the substrate concentration was a curve with saturation. At neutral pH the K(m) value of the mitochondrial "succinate oxidase" was fivefold less than that of the cellular "succinate oxidase". O-Palmitoyl-L-malate, not penetrating across the plasma membrane, completely inhibited cell respiration in the presence of succinate but not glucose or pyruvate. The linear inhibition in Dixon plots indicates that the rate of succinate oxidation is limited by its transport across the plasmalemma. O-Palmitoyl-L-malate and L-malate were competitive inhibitors (the K(i) values were 6.6 +/- 1.3 microM and 17.5 +/- 1.1 mM, respectively). The rate of succinate transport was also competitively inhibited by the malonate derivative 2-undecyl malonate (K(i) = 7.8 +/- 1.2 microM) but not phosphate. Succinate transport across the plasma membrane of S. cerevisiae is not coupled with proton transport, but sodium ions are necessary. The plasma membrane of S. cerevisiae is established to have a carrier catalyzing the transport of dicarboxylates (succinate and possibly L-malate and malonate).     

Converting the Yeast Arginine Can1 Permease to a Lysine Permease

Amino acid uptake in yeast cells is mediated by about 16 plasma membrane permeases, most of which belong to the amino acid-polyamine-organocation (APC) transporter family. These proteins display various substrate specificity ranges. For instance, the general amino acid permease Gap1 transports all amino acids, whereas Can1 and Lyp1 catalyze specific uptake of arginine and lysine, respectively. Although Can1 and Lyp1 have different narrow substrate specificities, they are close homologs. Here we investigated the molecular rules determining the substrate specificity of the H⁺-driven arginine-specific permease Can1. Using a Can1-Lyp1 sequence alignment as a guideline and a three-dimensional Can1 structural model based on the crystal structure of the bacterial APC family arginine/agmatine antiporter, we introduced amino acid substitutions liable to alter Can1 substrate specificity. We show that the single substitution T456S results in a Can1 variant transporting lysine in addition to arginine and that the combined substitutions T456S and S176N convert Can1 to a Lyp1-like permease. Replacement of a highly conserved glutamate in the Can1 binding site leads to variants (E184Q and E184A) incapable of any amino acid transport, pointing to a potential role for this glutamate in H⁺ coupling. Measurements of the kinetic parameters of arginine and lysine uptake by the wild-type and mutant Can1 permeases, together with docking calculations for each amino acid in their binding site, suggest a model in which residues at positions 176 and 456 confer substrate selectivity at the ligand-binding stage and/or in the course of conformational changes required for transport.     

AGP2 encodes the major permease for high affinity polyamine import in Saccharomyces cerevisiae

Polyamines play essential functions in many aspects of cell biology. Plasma membrane transport systems for the specific uptake of polyamines exist in most eukaryotic cells but have been very recently identified at the molecular level only in the parasite Leishmania. We now report that the high affinity polyamine permease in Saccharomyces cerevisiae is identical to Agp2p, a member of the yeast amino acid transporter family that was previously identified as a carnitine transporter. Deletion of AGP2 dramatically reduces the initial velocity of spermidine and putrescine uptake and confers strong resistance to the toxicity of exogenous polyamines, and transformation with an AGP2 expression vector restored polyamine transport in agp2delta mutants. Yeast mutants deficient in polyamine biosynthesis required >10-fold higher concentrations of exogenous putrescine to restore cell proliferation upon deletion of the AGP2 gene. Disruption of END3, a gene required for an early step of endocytosis, increased the abundance of Agp2p, an effect that was paralleled by a marked up-regulation of spermidine transport velocity. Thus, AGP2 encodes the first eukaryotic permease that preferentially uses spermidine over putrescine as a high affinity substrate and plays a central role in the uptake of polyamines in yeast.     

Expression of the cloned uracil permease gene of Saccharomyces cerevisiae in a heterologous membrane

A piece of DNA of the yeast Saccharomyces cerevisiae complementing the uracil permease gene was introduced into a plasmid able to replicate autonomously in Schizosaccharomyces pombe. A strain of S. pombe lacking uracil transport activity was transformed with this new plasmid carrying the gene of S. cerevisiae. The behaviour of the transformant shows not only an expression of the uracil permease gene in the heterologous membrane but also that the transport of uracil is active and coupled to the energy furnishing system of the heterologous host.