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.     

MAL11 and MAL61 encode the inducible high-affinity maltose transporter of Saccharomyces cerevisiae.

We have investigated the transport of maltose in a genetically defined maltose-fermenting strain of Saccharomyces cerevisiae carrying the MAL1 locus. Two kinetically different systems were identified: a high-affinity transporter with a Km of 4 mM and a low-affinity transporter with a Km of 70 to 80 mM. The high-affinity maltose transporter is maltose inducible and is encoded by the MAL11 (and/or MAL61) gene of the MAL1 (and/or MAL6) locus. The low-affinity maltose transporter is expressed constitutively and is not related to MAL11 and/or MAL61. Both maltose transporters are subject to glucose-induced inactivation.     

Clustering of MAL genes in Hansenula polymorpha: cloning of the maltose permease gene and expression from the divergent intergenic region between the maltose permease and maltase genes

Hansenula polymorpha uses maltase to grow on maltose and sucrose. Inspection of genomic clones of H. polymorpha showed that the maltase gene HPMAL1 is clustered with genes corresponding to Saccharomyces cerevisiae maltose permeases and MAL activator genes orthologues. We sequenced the H. polymorpha maltose permease gene HPMAL2 of the cluster. The protein (582 amino acids) deduced from the HPMAL2 gene is predicted to have eleven transmembrane domains and shows 39-57% identity with yeast maltose permeases. The identity of the protein is highest with maltose permeases of Debaryomyces hansenii and Candida albicans. Expression of the HPMAL2 in a S. cerevisiae maltose permease-negative mutant CMY1050 proved functionality of the permease protein encoded by the gene. HPMAL1 and HPMAL2 genes are divergently positioned similarly to maltase and maltose permease genes in many yeasts. A two-reporter assay of the expression from the HPMAL1-HPMAL2 intergenic region showed that expression of both genes is coordinately regulated, repressed by glucose, induced by maltose, and that basal expression is higher in the direction of the permease gene.     

The Maltose Permease Encoded by the Mal61 Gene of Saccharomyces Cerevisiae Exhibits Both Sequence and Structural Homology to Other Sugar Transporters

The MAL61 gene of Saccharomyces cerevisiae encodes maltose permease, a protein required for the transport of maltose across the plasma membrane. Here we report the nucleotide sequence of the cloned MAL61 gene. A single 1842 bp open reading frame is present within this region encoding the 614 residue putative MAL61 protein. Hydropathy analysis suggests that the secondary structure consists of two blocks of six transmembrane domains separated by an approximately 71 residue intracellular region. The N-terminal and C-terminal domains of 100 and 67 residues in length, respectively, also appear to be intracellular. Significant sequence and structural homology is seen between the MAL61 protein and the Saccharomyces high-affinity glucose transporter encoded by the SNF3 gene, the Kluyveromyces lactis lactose permease encoded by the LAC12 gene, the human HepG2 glucose transporter and the Escherichia coli xylose and arabinose transporters encoded by the xylE and araE genes, indicating that all are members of a family of sugar transporters and are related either functionally or evolutionarily. A mechanism for glucose-induced inactivation of maltose transport activity is discussed.     

A zinc finger protein from Candida albicans is involved in sucrose utilization.

A sucrose-inducible alpha-glucosidase activity that hydrolyzes sucrose in Candida albicans has been demonstrated previously. The enzyme is assayable in whole cells and was inhibited by both sucrose and maltose. A C. albicans gene (CASUC1) that affects sucrose utilization and alpha-glucosidase activity was cloned by expression in a Saccharomyces cerevisiae suc2 mutant (2102) devoid of invertase genes. CASUC1 enabled the S. cerevisiae mutant to utilize both sucrose and maltose. DNA sequence analysis revealed that CASUC1 encodes a putative zinc finger-containing protein with 28% identity to a maltose-regulatory gene (MAL63) of S. cerevisiae. The gene products of CASUC1 and MAL63 are approximately the same size (501 and 470 amino acids, respectively), and each contains a single zinc finger located at the N terminus. The zinc fingers of CASUC1 and MAL63 comprise six conserved cysteines (C6 zinc finger) and are of the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaavariable-Cys-Xaa2-Cys-+ ++Xaa6-Cys (where Xaan indicates a stretch of the indicated number of any amino acids). Both contain five amino acids in the variable region. CASUC1 also complemented the maltose utilization defect of an S. cerevisiae mutant (TCY-137) containing a defined mutation in a maltose-regulatory gene. The sucrose utilization defect of type II Candida stellatoidea, a sucrase-negative mutant of C. albicans, was corrected by CASUC1. Determinations of alpha-glucosidase activity in whole cells revealed that activity was restored in transformants cultivated on either sucrose or maltose. To our knowledge, this is the first zinc finger-encoding gene, as well as the first putative regulatory gene, to be identified in C. albicans.     

Primary structure of the maltose-permease-encoding gene of Saccharomyces carlsbergensis

The MAL6 locus of Saccharomyces consists of a cluster of at least three genes: MAL6R encodes a positively acting regulatory protein; MAL6S encodes maltase; and MAL6T encodes maltose permease. A MAL6 Eco RI fragment, E1, that encompasses most of the MAL6T gene except for the first 90 bp of the ORF at its 5' end (sequenced previously), was cloned into a pGEM-Blue vector. Sequential deletions were generated and then sequenced. The MAL6T gene has a putative ORF of 1845 bp. The amino acid composition and sequence of the deduced protein shows that it is highly hydrophobic and has a size of 68.2 kDa. Computer-generated hydropathy profiles suggest that the MAL6T protein may have up to nine membrane-spanning regions. Generation of functional fusions of the MAL6T promoter region to Escherichia coli lacZ-containing vectors indicates that sequences in the intergenic region are responsible for the induction of MAL6T by maltose and for its carbon catabolite repression. We also demonstrated the suitability of E. coli lacZ as a reporter gene for promoter activity studies in yeast.     

Isolation and characterization of maltose non utilizing (mnu) mutants mapping outside the MAL1 locus in Saccharomyces cerevisiae

The MAL1 locus of Saccharomyces cerevisiae comprises three genes necessary for maltose utilization. They include regulatory, maltose transport and maltase genes designated MAL1R, MAL1T and MAL1S respectively. Using a MAL1 strain transformed with an episomal, multicopy plasmid carrying the MAL2 locus, five recessive and one dominant mutant unable to grow on maltose, but still retaining a functional MAL1 locus were isolated. All the mutants could use glycerol, ethanol, raffinose and sucrose as a sole carbon source; expression of the maltase and maltose permease genes was severely and coordinately reduced. Only the dominant mutant failed to accumulate the MAL1R mRNA.     

Mutational analysis of the MAL1 locus of Saccharomyces: identification and functional characterization of three genes

Summary Fermentation of maltose by Saccharomyces strains depends on the presence of any one of five unlinked MAL loci (MAL1, MAL2, MAL3, MAL4 or MAL6). Earlier mutational analyses of MAL2 and MAL6 containing strains have identified a single complementation group at each of these two loci. However complementation analysis between naturally occurring Mal^– Saccharomyces strains isolated from the wild demonstrated the presence of two complementation groups (designated MALp and MALg) at the MAL1, MAL3 and MAL6 loci. The available evidence suggests that the MALp gene is functionally equivalent to the complementation group identified by mutational analysis at the MAL6 locus and that this gene encodes a protein involved in the regulation of the coordinate induction of both maltase and maltose permease synthesis.In this paper we report the isolation, in a well characterized MAL1 strain, of 47 mutants unable to ferment maltose. All the mutants, with one exception, map at the MAL1 locus. These mal1 mutants, except for one, are recessive to MAL1 and fall into two major complementation groups. Evidence is presented that these two classes of mutants identify both a gene involved in the regulation of maltose fermentation (MAL1R) and a gene involved in maltose transport (MAL1T). We also report here the isolation of a temperature sensitive maltose nonfermenting mutant mapping at the MAL1 locus identifying a third gene (MAL1S) at this locus. The maltase synthesized by this mutant, when assayed in cell-free extracts, is significantly more thermolabile than the wild type enzyme. Our findings demonstrate that MAL1 is a complex locus comprising at least three genes: MAL1R, a gene involved in the coordinate regulation of the synthesis of maltase and maltose transport; MAL1T, a gene encoding a component of the maltose transport system; and MAL1S, a likely candidate for the structural gene for maltase.     

Overexpression of Mal61p in Saccharomyces cerevisiae and characterization of maltose transport in artificial membranes.

For maltose uptake in Saccharomyces cerevisiae, multiple kinetic forms of transport as well as inhibition of transport by high concentrations of maltose at the trans side of the plasma membrane have been described. Most of these studies were hampered by a lack of genetically well-defined mutants and/or the lack of an artificial membrane system to study translocation catalysis in vitro. A genetically well-defined S. cerevisiae strain lacking the various MAL loci was constructed by gene disruption. Expression of the maltose transport protein (Mal61p) was studied by using various plasmid vectors that differed in copy number and/or type of promoter. The expression levels were quantitated by immunoblotting with antibodies generated against the N-terminal half of Mal61p. The levels of expression as well as the initial uptake rates were increased 20-fold compared with those in a yeast strain carrying only one chromosomal MAL locus. Similar results were obtained when the transport activities were compared in hybrid membranes of the corresponding strains. To generate a proton motive force, isolated membranes were fused with liposomes containing cytochrome c oxidase as a proton pump. Fusion was achieved by a cycle of freeze-thawing, after which the hybrid membranes were passed through a filter with a defined pore size to obtain unilamellar membrane vesicles. Proton motive force-driven maltose uptake, maltose efflux down the concentration gradient, and equilibrium exchange of maltose in the hybrid membranes vesicles have been analyzed. The data indicate that maltose transport by the maltose transporter is kinetically monophasic and fully reversible under all conditions tested.     

Maltotriose utilization by industrial Saccharomyces strains: characterization of a new member of the alpha-glucoside transporter family

Maltotriose utilization by Saccharomyces cerevisiae and closely related yeasts is important to industrial processes based on starch hydrolysates, where the trisaccharide is present in significant concentrations and often is not completely consumed. We undertook an integrated study to better understand maltotriose metabolism in a mixture with glucose and maltose. Physiological data obtained for a particularly fast-growing distiller's strain (PYCC 5297) showed that, in contrast to what has been previously reported for other strains, maltotriose is essentially fermented. The respiratory quotient was, however, considerably higher for maltotriose (0.36) than for maltose (0.16) or glucose (0.11). To assess the role of transport in the sequential utilization of maltose and maltotriose, we investigated the presence of genes involved in maltotriose uptake in the type strain of Saccharomyces carlsbergensis (PYCC 4457). To this end, a previously constructed genomic library was used to identify maltotriose transporter genes by functional complementation of a strain devoid of known maltose transporters. One gene, clearly belonging to the MAL transporter family, was repeatedly isolated from the library. Sequence comparison showed that the novel gene (designated MTY1) shares 90% and 54% identity with MAL31 and AGT1, respectively. However, expression of Mty1p restores growth of the S. cerevisiae receptor strain on both maltose and maltotriose, whereas the closely related Mal31p supports growth on maltose only and Agt1p supports growth on a wider range of substrates, including maltose and maltotriose. Interestingly, Mty1p displays higher affinity for maltotriose than for maltose, a new feature among all the alpha-glucoside transporters described so far.     

Repeated Family of Genes Controlling Maltose Fermentation in Saccharomyces carlsbergensis

Maltose fermentation in Saccharomyces spp. requires the presence of any one of five unlinked genes: MAL1, MAL2, MAL3, MAL4, or MAL6. Although the genes are functionally equivalent, their natures and relationships to each other are not known. At least three proteins are necessary for maltose fermentation: maltase, maltose permease, and a regulatory protein. The MAL genes may code for one or more of these proteins. Recently a DNA fragment containing a maltase structural gene has been cloned from a MAL6 strain, CB11, to produce plasmid pMAL9-26. We have conducted genetic and physical analyses of strain CB11. The genetic analysis has demonstrated the presence of two cryptic MAL genes in CB11, MAL1g and MAL3g (linked to MAL1 and to MAL3, respectively), in addition to the MAL6 locus. The physical analysis, which used a subclone of plasmid pMAL9-26 as a probe, detected three HindIII genomic fragments with homology to the probe. Each fragment was shown to be linked to one of the MAL loci genetically demonstrated to be present in CB11. Our results indicate that the cloned maltase structural gene in plasmid pMAL9-26 is linked to MAL6. Since the MAL6 locus has previously been shown to contain a regulatory gene, the MAL6 locus must be a complex locus containing at least two of the factors needed for maltose fermentation: the structural gene for maltase and the maltase regulatory protein. The absence of other fragments which hybridize to the MAL6-derived probe shows that either MAL2 and MAL4 are not related to MAL6, or the DNA corresponding to these genes is absent from the MAL6 strain CB11.     

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.     

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.     

Organization of the MAL loci of Saccharomyces. Physical identification and functional characterization of three genes at the MAL6 locus

Summary We have physically and functionally identified three genes at the MAL6 locus of Saccharomyces carlsbergensis. Using multicopy yeast plasmid vectors, we have subcloned various segments of the entire MAL6 locus. The functional characterization of the MAL6 subcloned regions was determined by (1) analyzing biochemically the levels of MAL-encoded proteins (maltase [-D-glucosidase, E.C. 3.2.1.20] and maltose transport protein) in cells transformed with various MAL6 subclones, and (2) testing the ability of the subclones to complement the maltose fermentation defects of well characterized Mal^– mutants in the highly homologous MAL1 locus. The physical homology between MAL6 and MAL1 is in part demonstrated by the gene disruption of MAL1 using subcloned MAL6 DNA sequences. The results demonstrate that the MAL6 locus is a complex of at least three genes: MAL6R, MAL6T and MAL6S. These genes specify, respectively, a regulatory function, a maltose transport activity (presumably the maltose permease) and the structural gene for maltase. The functional organization of the MAL6 locus is thus identical to that which we had previously determined by mutational analysis for the MAL1 locus.