Identification of a second trans-acting gene controlling maltose fermentation in Saccharomyces carlsbergensis.

Maltose fermentation in Saccharomyces spp. requires the presence of a dominant MAL locus. The MAL6 locus has been cloned and shown to encode the structural genes for maltose permease (MAL61), maltase (MAL62), and a positively acting regulatory gene (MAL63). Induction of the MAL61 and MAL62 gene products requires the presence of maltose and the MAL63 gene. Mutations within the MAL63 gene produce nonfermenting strains unable to induce the two structural gene products. Reversion of these mal63 nonfermenters to maltose fermenters nearly always leads to the constitutive expression of maltase and maltose permease, and constitutivity is always linked to MAL6. We demonstrated that for one such revertant, strain C2, constitutivity did not require the MAL63 gene, since deletion disruption of this gene did not affect the constitutive expression of the structural genes. In addition, constitutivity was trans acting. Deletion disruption of the MAL6-linked structural genes for maltase and maltose permease in this strain did not affect the constitutive expression of a second, unlinked maltase structural gene. We isolated new maltose-fermenting revertants of a nonfermenting strain which carried a deletion disruption of the MAL63 gene. All 16 revertants isolated expressed maltase constitutively. In one revertant studied in detail, strain R10, constitutive expression was demonstrated to be linked to MAL6, semidominant, trans acting, and residing outside the MAL63-MAL61-MAL62 genes. From these studies we propose the existence of a second trans-acting regulatory gene at the MAL6 locus. We call this new gene MAL64. We mapped the MAL64 gene 2.3 centimorgans to the left of MAL63. The role of the MAL64 gene product in maltose fermentation is discussed.     

The Constitutive, Glucose-Repression-Insensitive Mutation of the Yeast MAL4 Locus Is an Alteration of the MAL43 Gene

Mutations resulting in constitutive production of maltase have been identified at each of the five MAL loci of Saccharomyces yeasts. Here we examine a dominant constitutive, glucose-repression-insensitive allele of the MAL4 locus (MAL4-C). Our results demonstrate that MAL4-C is an alteration in the MAL43 gene, which encodes the positive regulator of the MAL structural genes, and that its product is trans-acting. The MAL43 gene from the MAL4-C strain was cloned and integrated into a series of nonfermenting strains lacking a functional regulatory gene but carrying copies of the maltose permease and maltase structural genes. Expression of the maltase structural gene was both constitutive and insensitive to glucose repression in these transformants. The MAL4-C allele also results in constitutive expression of the unlinked MAL12 gene (encoding maltase) in this strain. In addition, the cloned MAL43 gene was shown to be dominant to the wild-type MAL63 gene. We also show that most of the glucose repression insensitivity of strains carrying the MAL4-C allele results from alteration of MAL43.     

Identification of the upstream activating sequence of MAL and the binding sites for the MAL63 activator of Saccharomyces cerevisiae.

Maltose fermentation in Saccharomyces species requires the presence of at least one of five unlinked MAL loci: MAL1, MAL2, MAL3, MAL4, and MAL6. Each of these loci consists of a complex of genes involved in maltose metabolism; the complex includes maltase, a maltose permease, and an activator of these genes. At the MAL6 locus, the activator is encoded by the MAL63 gene. While the MAL6 locus has been the subject of numerous studies, the binding sites of the MAL63 activator have not been determined. In this study, we used Escherichia coli extracts containing the MAL63 protein to define the binding sites of the MAL63 protein in the divergently transcribed MAL61-62 promotor. When a DNA fragment containing these sites was placed upstream of a CYC1-lacZ gene, maltose induced beta-galactosidase. These sites therefore constitute an upstream activating sequence for the MAL genes.     

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.     

Characterization of AGT1 encoding a general α-glucoside transporter from Saccharomyces

Molecular genetic analysis is used to characterize the AGT1 gene encoding an α-glucoside transporter. AGT1 is found in many Saccharomyces cerevisiae laboratory strains and maps to a naturally occurring, partially functional allele of the MAL1 locus. Agt1p is a highly hydrophobic, postulated integral membrane protein. It is 57% identical to Mal61p, the maltose permease encoded at MAL6 , and is also a member of the 12 transmembrane domain superfamily of sugar transporters. Like Mal61p, Agt1p is a high-affinity, maltose/proton symporter, but Mal61p is capable of transporting only maltose and turanose, while Agt1p transports these two α-glucosides as well as several others including isomaltose, α-methylglucoside, maltotriose, palatinose, trehalose and melezitose. AGT1 expression is maltose inducible and induction is mediated by the Mal-activator. The sequence of the upstream region of AGT1 is identical to that of the maltose-inducible MAL61 gene over a 469 bp region containing the UAS_MAL but the 315 bp sequence immediately upstream of AGT1 shows no significant homology to the sequence immediately upstream of MAL61 . The evolutionary origin of the MAL1 allele to which AGT1 maps and the relationship of AGT1 to other α-glucoside fermentation genes is discussed.     

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.     

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.     

Control of maltase synthesis in yeast

Maltose fermentation in Saccharomyces species requires the presence of at least one of five unlinked MAL loci: MAL1, MAL2, MAL3, MAL4 and MAL6. Each MAL locus is complex consisting of at least three genes: a trans-acting activator, a maltose permease, and maltase. All the MAL loci show homology to each other both at the sequence level as determined by Southern transfer analysis and at the functional level as determined by complementation. We describe the organization of the MAL loci in yeast and the basic features of their regulation. The analysis of MAL has contributed to our understanding of the evolution of multigenic families, the global integration of carbohydrate metabolism, and gene regulation.     

Genetic Analysis of Haploids from Industrial Strains of Baker's Yeast

Strains of baker's yeast conventionally used by the baking industry in Japan were tested for the ability to sporulate and produce viable haploid spores. Three isolates which possessed the properties of baker's yeasts were obtained from single spores. Each strain was a haploid, and one of these strains, YOY34, was characterized. YOY34 fermented maltose and sucrose, but did not utilize galactose, unlike its parental strain. Genetic analysis showed that YOY34 carried two MAL genes, one functional and one cryptic; two SUC genes; and one defective gal gene. The genotype of YOY34 was identified as MATalpha MAL1 MAL3g SUC2 SUC4 gall. The MAL1 gene from this haploid was constitutively expressed, was dominant over other wild-type MAL tester genes, and gave a weak sucrose fermentation. YOY34 was suitable for both bakery products, like conventional baker's yeasts, and for genetic analysis, like laboratory strains.     

Constitutive Mutations of the Saccharomyces Cerevisiae Mal-Activator Genes Mal23, Mal43, Mal63, and Mal64

We report the sequence of several MAL-activator genes, including inducible, constitutive, and noninducible alleles of MAL23, MAL43, MAL63, and mal64. Constitutive alleles of MAL23 and MAL43 vary considerably from inducible alleles in their C-terminal domain, with many of the alterations clustered and common to both alleles. The 27 alterations from residues 238-461 of Mal43-C protein are sufficient for constitutivity, but the minimal number of alterations needed for the constitutive phenotype could not be determined. The sequence of mal64, a nonfunctional homologue of MAL63, revealed that Mal64p is 85% identical to Mal63p. Two mutations that activate mal64 and cause constitutivity are nonsense mutations resulting in truncated proteins of 306 and 282 residues. We conclude that the C-terminal region of the MAL-activator, from residues 283-470, contains a maltose-responsive negative regulatory domain, and that extensive mutation or deletion of the entire region causes loss of the negative regulatory function. Additionally, certain sequence elements in the region appear to be necessary for efficient induction of the full-length Mal63 activator protein. These studies highlight the role of ectopic recombination as an important mechanism of mutagenesis of the telomere-associated family of MAL loci.     

Removal of a Mig1p Binding Site Converts a Mal63 Constitutive Mutant Derived by Interchromosomal Gene Conversion to Glucose Insensitivity

Maltose fermenting strains of Saccharomyces cerevisiae have one or more complex loci called MAL. Each locus comprises at least three genes: MALx1 encodes maltose permease, MALx2 encodes maltase, and MALx3 encodes an activator of MALx1 and MALx2 (x denotes one of five MAL loci, with x = 1, 2, 3, 4, or 6). The MAL43(c) allele is constitutive and relatively insensitive to glucose repression. To understand better this unique phenotype of MAL43(c), we have isolated several MAL63(c) constitutive mutants from a MAL6 strain. All constitutive mutants remain glucose repressible, and all have multiple amino acid substitutions in the C-terminal region, now making this region of Mal63(c)p similar to that of Mal43(c)p. These changes have been generated by gene conversion, which transfers DNA from the telomeres of chromosome II and chromosome III or XVI to chromosome VIII (MAL6). The removal of a Mig1p binding site from the MAL63(c) promoter leads to a loss of glucose repression, imitating the phenotype of MAL43(c). Conversely, addition of a Mig1p binding site to the promoter of MAL43(c) converts it to glucose sensitivity. Mig1p modulation of Mal63p and Mal43p expression therefore plays a substantial role in glucose repression of the MAL genes.     

The Naturally Occurring Alleles of Mal1 in Saccharomyces Species Evolved by Various Mutagenic Processes Including Chromosomal Rearrangement

In order for a yeast strain to ferment maltose it must contain any one of the five dominant MAL loci. Each dominant MAL locus thus far analyzed contains three genes: GENE 1, encoding maltose permease, GENE 2 encoding maltase and GENE 3 encoding a positive trans-acting regulatory protein. In addition to these dominant MAL loci, several naturally occurring, partially functional alleles of MAL1 and MAL3 have been identified. Here, we present genetic and molecular analysis of the three partially functional alleles of MAL1: the MAL1p allele which can express only the MAL activator; the MAL1 g allele which can express both a maltose permease and maltase; and the mal1(0) allele which can express only maltase. Based on our results, we propose that the MAL1p, MAL1g and mal1(0) alleles evolved from the dominant MAL1 locus by a series of rearrangements and/or deletions of this yeast telomere-associated locus as well as by other mutagenic processes of gene inactivation. One surprising finding is that the MAL1g-encoded maltose permease exhibits little sequence homology to the MAL1-encoded maltose permease though they appear to be functionally homologous.     

Comparative genetics of yeasts. XXII. The determination of alpha-methylglucoside fermentation by the maltose genes MAL6c2 and malx in the offspring of Saccharomyces cerevisiae N. C. Y. C. 74 strain

In offsprings of N.C.Y.C. 74, maltose regulatory constitutive MAL6C2 mutation controls alpha-methylglucoside (alpha-mgl) fermentation in the presence of MALx. MAL6C2 MALx system described by ten Berge et al, is analogous in function (polymeric interaction) to, at least, one MGLa gene from the system of complementary alpha-mgl genes MGLa MGLb MGLc identified by ten Berge. Suppressor malx mutation inhibits both the maltose and alpha-mgl activity of MAL6C2 allele. A brief review on participation of maltose genes in alpha-mgl fermentation is presented.