Molecular Analysis of Maltotriose Active Transport and Fermentation by Saccharomyces cerevisiae Reveals a Determinant Role for the AGT1 Permease

Incomplete and/or sluggish maltotriose fermentation causes both quality and economic problems in the ale-brewing industry. Although it has been proposed previously that the sugar uptake must be responsible for these undesirable phenotypes, there have been conflicting reports on whether all the known α-glucoside transporters in Saccharomyces cerevisiae (MALx1, AGT1, and MPH2 and MPH3 transporters) allow efficient maltotriose utilization by yeast cells. We characterized the kinetics of yeast cell growth, sugar consumption, and ethanol production during maltose or maltotriose utilization by several S. cerevisiae yeast strains (both MAL constitutive and MAL inducible) and by their isogenic counterparts with specific deletions of the AGT1 gene. Our results clearly showed that yeast strains carrying functional permeases encoded by the MAL21, MAL31, and/or MAL41 gene in their plasma membranes were unable to utilize maltotriose. While both high- and low-affinity transport activities were responsible for maltose uptake from the medium, in the case of maltotriose, the only low-affinity (K_m, 36 ± 2 mM) transport activity was mediated by the AGT1 permease. In conclusion, the AGT1 transporter is required for efficient maltotriose fermentation by S. cerevisiae yeasts, highlighting the importance of this permease for breeding and/or selection programs aimed at improving sluggish maltotriose fermentations.     

Protein phosphatase type-1 regulatory subunits Reg1p and Reg2p act as signal transducers in the glucose-induced inactivation of maltose permease in Saccharomyces cerevisiae

The REG1 gene encodes a regulatory subunit of the type-1 protein phosphatase (PP1) Glc7 in Saccharomyces cerevisiae, which directs the catalytic subunit to substrates involved in glucose repression. Loss of REG1 relieves glucose repression of many genes, including the MAL structural genes that encode the maltose fermentation enzymes. In this report, we explore the role of Reg1p and its homolog Reg2p in glucose-induced inactivation of maltose permease. Glucose stimulates the proteolysis of maltose permease and very rapid loss of maltose transport activity – more rapid than can be explained by loss of the permease protein alone. In a reg1Δ strain we observe a significantly reduced rate of glucose-induced proteolysis of maltose permease, and the rapid loss of maltose transport activity does not occur. Instead, surprisingly, the slow rate of proteolysis of maltose permease is accompanied by an increase in maltose transport activity. Loss of Reg2p modestly reduces the rates of both glucose-induced proteolysis of maltose permease and inactivation of maltose transport activity. Overexpression of Reg2p in a reg1Δ strain suppresses the effect on maltose permease proteolysis and partially restores the inactivation of maltose transport activity, but does not affect the insensitivity of MAL gene expression to repression by glucose observed in this strain. Thus, protein phosphatase type-1 (Glc7p-Reg1p and Glc7p-Reg2p) plays a role in transduction of the glucose signal during glucose-induced proteolysis of maltose permease, but only Glc7p-Reg1p is involved in glucose-induced inactivation of maltose transport activity and glucose repression of MAL gene expression. Overexpression of REG1 partially restores proteolysis of maltose permease in a grr1Δ strain, which lacks glucose signaling, but does not rescue rapid inactivation of maltose transport activity or sensitivity to glucose repression. A model for the role of Reg1p and Reg2p in glucose signaling pathways is discussed. We also uncovered a previously unrecognized G2/M delay in the grr1Δ but not the reg1Δ strains, and this delay is suppressed by REG1 overexpression. The G1/S delay seen in grr1Δ mutants is slightly suppressed as well, but REG1 overexpression does not suppress other grr1Δ phenotypes such as insensitivity to glucose repression. Received: 21 October 1999 / Accepted: 28 December 1999     

Maltotriose Utilization by Industrial Saccharomyces Strains: Characterization of a New Member of the α-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 α-glucoside transporters described so far.     

Agp2, a Member of the Yeast Amino Acid Permease Family,Positively Regulates Polyamine Transport at the Transcriptional Level

Agp2 is a plasma membrane protein of the Saccharomyces cerevisiae amino acid transporter family, involved in high-affinity uptake of various substrates including L-carnitine and polyamines. The discovery of two high affinity polyamine permeases, Dur3 and Sam3, prompted us to investigate whether Agp2 directly transports polyamines or acts instead as a regulator. Herein, we show that neither dur3Δ nor sam3Δ single mutant is defective in polyamine transport, while the dur3Δ sam3Δ double mutant exhibits a sharp decrease in polyamine uptake and an increased resistance to polyamine toxicity similar to the agp2Δ mutant. Studies of Agp2 localization indicate that in the double mutant dur3Δ sam3Δ, Agp2-GFP remains plasma membrane-localized, even though transport of polyamines is strongly reduced. We further demonstrate that Agp2 controls the expression of several transporter genes including DUR3 and SAM3, the carnitine transporter HNM1 and several hexose, nucleoside and vitamin permease genes, in addition to SKY1 encoding a SR kinase that positively regulates low-affinity polyamine uptake. Furthermore, gene expression analysis clearly suggests that Agp2 is a strong positive regulator of additional biological processes. Collectively, our data suggest that Agp2 might respond to environmental cues and thus regulate the expression of several genes including those involved in polyamine transport.     

Multiple α-Glucoside Transporter Genes in Brewer’s Yeast

Maltose and maltotriose are the two most abundant fermentable sugars in brewer’s wort, and the rate of uptake of these sugars by brewer’s yeast can have a major impact on fermentation performance. In spite of this, no information is currently available on the genetics of maltose and maltotriose uptake in brewing strains of yeast. In this work, we studied 30 brewing strains of yeast (5 ale strains and 25 lager strains) with the aim of examining the alleles of maltose and maltotriose transporter genes contained by them. To do this, we hybridized gene probes to chromosome blots. Studies performed with laboratory strains have shown that maltose utilization is conferred by any one of five unlinked but highly homologous MAL loci (MAL1 to MAL4 and MAL6). Gene 1 at each locus encodes a maltose transporter. All of the strains of brewer’s yeast examined except two were found to contain MAL11 and MAL31 sequences, and only one of these strains lacked MAL41. MAL21 was not present in the five ale strains and 12 of the lager strains. MAL61 was not found in any of the yeast strains. In three of the lager strains, there was evidence that MAL transporter gene sequences occurred on chromosomes other than those known to carry MAL loci. Sequences corresponding to the AGT1 gene, which encodes a transporter of several α-glucosides, including maltose and maltotriose, were detected in all but one of the yeast strains. Homologues of AGT1 were identified in three of the lager strains, and two of these homologues were mapped, one to chromosome II and the other to chromosome XI. AGT1 appears to be a member of a family of closely related genes, which may have arisen in brewer’s yeast in response to selective pressure.     

PmSUC3: characterization of a SUT2/SUC3-type sucrose transporter from Plantago major

Higher plants possess medium-sized gene families that encode plasma membrane-localized sucrose transporters. For several plant species, it has been shown that at least one of these genes (e.g., AtSUC3 in Arabidopsis and LeSUT2 in tomato) differs from all other family members in several features, such as the length of the open reading frame, the number of introns, and the codon usage bias. For these reasons, and because two of these proteins did not rescue a yeast mutant defective in sucrose utilization, it had been speculated that this subgroup of transporters might have sensor functions. Here, we describe the detailed functional characterization and cellular localization of PmSUC3, the orthologous transporter from the Plantago major transporter family. The PmSUC3 protein is localized in the sieve elements of the Plantago phloem and mediates the energy-dependent transport of sucrose and maltose. In contrast to the situation in solanaceous plants, PmSUC3 is not colocalized with PmSUC2, the source-specific, phloem-loading sucrose transporter of Plantago. Moreover, PmSUC3 also was identified in sieve elements of sink leaves and in several nonphloem cells and tissues. Arguments for and against a potential sensor function for this type of sucrose transporter are presented, and the role of this type of transporter in the regulation of sucrose fluxes is discussed.     

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.     

Evidence for the Transport of Maltose by the Sucrose Permease,CscB, of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The purpose of this study was to examine the sugar recognition and transport properties of the sucrose permease (CscB), a secondary active transporter from Escherichia coli. We tested the hypothesis that maltose transport is conferred by the wild-type CscB transporter. Cells of E. coli HS4006 harboring pSP72/cscB were red on maltose MacConkey agar indicator plates. We were able to measure “downhill” maltose transport and establish definitive kinetic behavior for maltose entry in such cells. Maltose was an effective competitor of sucrose transport in cells with CscB, suggesting that the respective maltose and sucrose binding sites and translocation pathways through the CscB channel overlap. Accumulation (“uphill” transport) of maltose by cells with CscB was profound, demonstrating active transport of maltose by CscB. Sequencing of cscB encoded on plasmid pSP72/cscB used in cells for transport studies indicate an unaltered primary CscB structure, ruling out the possibility that mutation conferred maltose transport by CscB. We conclude that maltose is a bona fide substrate for the sucrose permease of E. coli. Thus, future studies of sugar binding, transport, and permease structure should consider maltose, as well as sucrose. Yang Peng and Sanath Kumar contributed equally to this paper.     

Cloning and characterization of an aromatic amino acid and leucine permease of Penicillium chrysogenum

The gene encoding the amino acid permease ArlP (Aromatic and leucine Permease) was isolated from the filamentous fungus Penicillium chrysogenum after PCR using degenerated oligonucleotides based on conserved regions of fungal amino acid permeases. The cDNA clone was used for expression of the permease in Saccharomyces cerevisiae M4054, which is defective in the general amino acid permease Gap1. Upon overexpression, an increase in the uptake of l-tyrosine, l-phenylalanine, l-tryptophan and l-leucine was observed. Further competition experiments indicate that ArlP recognizes neutral and aromatic amino acids with an unbranched β-carbon atom.     

A PEST-like sequence in the N-terminal cytoplasmic domain of Saccharomyces maltose permease is required for glucose-induced proteolysis and rapid inactivation of transport activity

Maltose permease is required for maltose transport into Saccharomyces cells. Glucose addition to maltose-fermenting cells causes selective delivery of this integral plasma membrane protein to the yeast vacuole via endocytosis for degradation by resident proteases. This glucose-induced degradation is independent of the proteasome but requires ubiquitin and certain ubiquitin conjugating enzymes. We used mutation analysis to identify target sequences in Mal61/HA maltose permease involved in its selective glucose-induced degradation. A nonsense mutation was introduced at codon 581, creating a truncated functional maltose permease. Additional missense mutations were introduced into the mal61/HA-581NS allele, altering potential phosphorylation and ubiquitination sites. No significant effect was seen on the rate of glucose-induced degradation of these mutant proteins. Deletion mutations were constructed, removing residues 2-30, 31-60, 61-90, and 49-78 of the N-terminal cytoplasmic domain, as well as a missense mutation of a dileucine motif. Results indicate that the proline-, glutamate-, aspartate-, serine-, and threonine-rich (PEST) sequence found in the N-terminal cytoplasmic domain, particularly residues 49-78, is required for glucose-induced degradation of Mal61/HAp and for the rapid glucose-induced inactivation of maltose transport activity. The decreased rate of glucose-induced degradation correlates with a decrease in the level of glucose-induced ubiquitination of the DeltaPEST mutant permease. In addition, newly synthesized mutant permease proteins lacking residues 49-78 or carrying an alteration in the dileucine motif, residues 69 and 70, are resistant to glucose-induced inactivation of maltose transport activity. This N-terminal PEST-like sequence is the target of both the Rgt2p-dependent and the Glc7p-Reg1p-dependent glucose signaling pathways.     

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.     

Enhancement of maltose utilisation by Saccharomyces cerevisiae in medium containing fermentable hexoses

Saccharomyces cerevisiae are unable to maintain high rates of fermentation during transition from catabolism of hexoses to maltose. This phenomenon, termed ‘maltose lag’, presents problems for the baking, brewing and distilling industries, which rely on yeast catabolism of mixtures of hexoses and maltose. Maltose utilisation requires the presence of maltose permease and α-glucosidase (maltase), encoded by MAL genes. Synthesis of these is induced by maltose and repressed by glucose. One strain of baker’s yeast used in this work exhibited a marked maltose lag, whereas a second strain exhibited a shorter lag during conversion from hexose to maltose metabolism. The extent of the lag was linked to the levels of maltose permease and maltase in cells at the time of inoculation into mixed sugar medium. This view is supported by results showing that pulsing yeast with maltose to induce expression of MAL genes prior to inoculation into mixed sugar medium, enhanced sugar fermentation. Maltose pulsing of yeasts could therefore be useful for enhancing some fermentations relevant to baking and other yeast industries. Received 24 December 1988/ Accepted in revised form 18 March 1999     

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.     

Structure of the multigene family of MAL loci in Saccharomyces

Summary Multigene families are a ubiquitous feature of eukaryotes; however, their presence in Saccharomyces is more limited. The MAL multigene family is comprised of five unlined loci, MAL1, MAL2, MAL3, MAL4 and MAL6, any one of which is sufficient for yeast to metabolize maltose. A cloned MAL6 locus was used as a probe to facilitate the cloning of the other four functional loci as well as two partially active alleles of MAL1. Each locus could be characterized as a cluster of three genes, MALR (regulatory), MALT (maltose transport or permease) and MALS (structural or maltase), encoded by a total of about 7 kb of DNA; however, homologous sequences at each locus extend beyond the coding regions. Our results indicate that there is extensive homology among the MAL loci, especially within their maltase genes. The greatest sequence diversity occurs in their regulatory gene regions. Southern cross analyses of the cloned MAL loci indicate a single duplication of the MAL6R-homologous sequences upstream of the MAL6R gene as well as an extensive duplication of more than 10 kb at the MAL3 locus. The large repeat at the MAL3 locus results in the presence of four copies of MAL3R-homologous sequences and two copies of MAL3T-homologous sequences at that locus. Two naturally occurring inactive alleles of MAL1 show a deletion or divergence of their MALR sequences. The significance of these repeats in the evolution of the MAL multigene family is discussed.     

A phloem-specific sucrose-H+ symporter from Plantago major L. supports the model of apoplastic phloem loading

In this paper the cloning of a full-length cDNA clone encoding the PmSUC2 sucrose-H⁺ symporter from Plantago major is described. This plant allows the simple preparation of vascular bundles from the basal regions of fully developed source leaves and thus a separation of vascular and non-vascular tissue. A cDNA library was constructed from poly(A)⁺ RNA isolated from vascular bundles and used for the subsequent cloning of cDNAs. The respective mRNA is specifically expressed in the vascular bundles as shown on Northern blots of total RNA from vascular and non-vascular tissues. The PmSUC2 protein has 12 putative transmembrane helices and is highly homologous to other plant sucrose transporters. Substrate specificity and energy dependence of the transporter encoded by this cDNA were determined by expression in baker's yeast Saccharomyces cerevisiae. The PmSUC2 protein catalyses the transport of sucrose into transgenic yeast cells. Invertase null mutants of yeast expressing PmSUC2 accumulate sucrose more than 200-fold. This transport was sensitive to uncouplers or SH-group inhibitors. Plasma membranes from yeast cells expressing the PmSUC2 protein were purified and fused to proteoliposomes containing cytochrome-c-oxidase. In this system sucrose is accumulated only when proton motive force is generated, indicating that PmSUC2 is a sucrose-H⁺ symporter. The apparent molecular weight of the PmSUC2 protein is 35 kDa on 10% SDS-polyacrylamide gels. The presented data strongly support the theory of phloem loading from the apoplastic space by a sucrose-H⁺ symporter.     

std1, a Gene Involved in Glucose Transport in Schizosaccharomyces pombe

A wild-type strain, Sp972 h⁻, of Schizosaccharomyces pombe was mutagenized with ethylmethanesulfonate (EMS), and 2-deoxyglucose (2-DOG)-resistant mutants were isolated. Out of 300 independent 2-DOG-resistant mutants, 2 failed to grow on glucose and fructose (mutants 3/8 and 3/23); however, their hexokinase activity was normal. They have been characterized as defective in their sugar transport properties, and the mutations have been designated as std1-8 and std1-23 (sugar transport defective). The mutations are allelic and segregate as part of a single gene when the mutants carrying them are crossed to a wild-type strain. We confirmed the transport deficiency of these mutants by [¹⁴C]glucose uptake. They also fail to grow on other monosaccharides, such as fructose, mannose, and xylulose, as well as disaccharides, such as sucrose and maltose, unlike the wild-type strain. Lack of growth of the glucose transport-deficient mutants on maltose revealed the extracellular breakdown of maltose in S. pombe, unlike in Saccharomyces cerevisiae. Both of the mutants are unable to grow on low concentrations of glucose (10 to 20 mM), while one of them, 3/23, grows on high concentrations (50 to 100 mM) as if altered in its affinity for glucose. This mutant (3/23) shows a lag period of 12 to 18 h when grown on high concentrations of glucose. The lag disappears when the culture is transferred from the log phase of its growth on high concentrations. These mutants complement phenotypically similar sugar transport mutants (YGS4 and YGS5) reported earlier by Milbradt and Hoefer (Microbiology 140:2617–2623, 1994), and the clone complementing YGS4 and YGS5 was identified as the only glucose transporter in fission yeast having 12 transmembrane domains. These mutants also demonstrate two other defects: lack of induction and repression of shunt pathway enzymes and defective mating.     

Two Distinct Pathways for Trehalose Assimilation in the Yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae can synthesize trehalose and also use this disaccharide as a carbon source for growth. However, the molecular mechanism by which extracellular trehalose can be transported to the vacuole and degraded by the acid trehalase Ath1p is not clear. By using an adaptation of the assay of invertase on whole cells with NaF, we showed that more than 90% of the activity of Ath1p is extracellular, splitting of the disaccharide into glucose. We also found that Agt1p-mediated trehalose transport and the hydrolysis of the disaccharide by the cytosolic neutral trehalase Nth1p are coupled and represent a second, independent pathway, although there are several constraints on this alternative route. First, the AGT1/MAL11 gene is controlled by the MAL system, and Agt1p was active in neither non-maltose-fermenting nor maltose-inducible strains. Second, Agt1p rapidly lost activity during growth on trehalose, by a mechanism similar to the sugar-induced inactivation of the maltose permease. Finally, both pathways are highly pH sensitive and effective growth on trehalose occurred only when the medium was buffered at around pH 5.0. The catabolism of trehalose was purely oxidative, and since levels of Ath1p limit the glucose flux in the cells, batch cultures on trehalose may provide a useful alternative to glucose-limited chemostat cultures for investigation of metabolic responses in yeast.