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For maltose fermentation, budding yeast Saccharomyces cerevisiae operates a mechanism that involves transporters (MALT), maltases (MALS) and regulators (MALR) collectively known as MAL genes. However, functional relevance of MAL genes during sake brewing process remains largely elusive, since sake yeast is cultured under glucose-rich condition achieved by the co-culture partner Aspergillus spp.. Here we isolated an ethyl methane sulfonate (EMS)-mutagenized sake yeast strain exhibiting enhanced maltose fermentation compared to the parental strain. The mutant carried a single nucleotide insertion that leads to the extension of the C-terminal region of a previously uncharacterized MALR gene YPR196W-2, which was renamed as MAL73. Introduction of the mutant allele MAL73L with extended C-terminal region into the parental or other sake yeast strains enhanced the growth rate when fed with maltose as the sole carbon source. In contrast, disruption of endogenous MAL73 in the sake yeasts decreased the maltose fermentation ability of sake yeast, confirming that the original MAL73 functions as a MALR. Importantly, the MAL73L-expressing strain fermented more maltose in practical condition compared to the parental strain during sake brewing process. Our data show that MAL73(L) is a novel MALR gene that regulates maltose fermentation, and has been functionally attenuated in sake yeast by single nucleotide deletion during breeding history. Since the MAL73L-expressing strain showed enhanced ability of maltose fermentation, MAL73L might also be a valuable tool for enhancing maltose fermentation in yeast in general., Competing Interests: TO, FO, HH and EO are employees of Suntory Global Innovation Center Ltd (SIC), a company affiliated to Suntory Holdings Ltd. SIC financially provides salaries of TO, FO, HH and EO and research materials of this study. There are no patents, products in development or marketed products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The maltose transporter gene is situated at the MAL locus, which consists of genes for a transporter, maltase, and transcriptional activator. Five unlinked MAL loci (MAL1, MAL2, MAL3, MAL4, and MAL6) constitute a gene family in Saccharomyces cerevisiae. The expression of the maltose transporter is induced by maltose and repressed by glucose. The activity of the maltose transporter is also regulated post-translationally; Mal61p is rapidly internalized from the plasma membrane and degraded by ubiquitin-mediated proteolysis in the presence of glucose. We found that S. cerevisiae strain ATCC20598 harboring MAL21 could grow in maltose supplemented with a non- assimilable glucose analogue, 2-deoxyglucose, whereas strain ATCC96955 harboring MAL61 and strain CB11 with MAL31 and AGT1 could not. These observations implied a Mal21p-specific resistance against glucose-induced degradation. Mal21p found in ATCC20598 has 10 amino acids, including Gly-46 and His-50, that are inconsistent with the corresponding residues in Mal61p. The half-life of Mal21p for glucose-induced degradation was 118 min when expressed using the constitutive TPI1 promoter, which was significantly longer than that of Mal61p (25 min). Studies with mutant cells that are defective in endocytosis or the ubiquitination process indicated that Mal21p was less ubiquitinated than Mal61p, suggesting that Mal21p remains on the plasma membrane because of poor susceptibility to ubiquitination. Mutational studies revealed that both residues Gly-46 and His-50 in Mal21p are essential for the full resistance of maltose transporters against glucose-induced degradation.

The Saccharomyces cerevisiae Put4 permease is significant for the transport of proline, alanine, and glycine. Put4p downregulation is counteracted by npi1 mutation that affects the cellular ubiquitination function. Here we describe mutant Put4 permeases, in which up to nine lysine residues in the cytoplasmic N-terminal domain have been replaced by arginine. The steady-state protein level of the mutant permease Put4-20p (Lys9, Lys34, Lys35, Lys60, Lys68, Lys71, Lys93, Lys105, Lys107 --> Arg) was largely higher compared to that of the wild-type Put4p, indicating that the N-terminal lysines can undergo ubiquitination and the subsequent degradation steps. Proline is the only amino acid that yeast assimilates with difficulty under standard brewing conditions. A lager yeast strain provided with Put4-20p was able to assimilate proline efficiently during beer fermentations. These results suggest possible industrial applications of the mutant Put4 permeases in improved fermentation systems for beer and other alcoholic beverages based on proline-rich fermentable sources.

The Saccharomyces cerevisiae branched-chain amino acid permease Bap2p plays a major role in leucine, isoleucine, and valine transport, and its synthesis is regulated transcriptionally. Bap2p undergoes a starvation-induced degradation depending upon ubiquitination and the functions of N- and C-terminal domains of Bap2p. Here we show that the N-terminal domain of Bap2p is phosphorylated in response to rapamycin treatment when both the N- and C-termini of Bap2p are fused to glutathione S-transferase. The phosphorylation is dependent on Ser/Thr kinase Npr1p. In npr1 cells, Bap2p becomes slightly more susceptible to the rapamycin-induced degradation, suggesting that Npr1p counteracts the degradation system for Bap2p.

The amino acid permease Bap2p in Saccharomyces cerevisiae mediates a major part of the uptake of leucine, isoleucine, and valine from media containing a preferred nitrogen source. Although the transcriptional controls of BAP2 have been well studied, the posttranslational down-regulation mechanisms for Bap2p have not been established. Here we show that Bap2p is subject to a starvation-induced degradation upon rapamycin treatment or cultivation with proline as the sole nitrogen source. The starvation-induced degradation of Bap2p was dependent on the cellular functions of ubiquitination and endocytosis. Down-regulation of the permease required the most probable ubiquitination sites, the lysine residues situated in the N-terminal 49 residues, as well as the C-terminal domain. Furthermore, when the N-terminal domain of Bap2p was fused to the general amino acid permease Gap1p, the resultant chimeric permease became susceptible to the starvation-induced degradation, indicating that the Bap2p N-terminus contains a determinant responsive to the starvation signals., (Copyright 2001 Academic Press.)

We found two types of branched-chain amino acid permease gene (BAP2) in the lager brewing yeast Saccharomyces pastorianus BH-225 and cloned one type of BAP2 gene (Lg-BAP2), which is identical to that of Saccharomyces bayanus (by-BAP2-1). The other BAP2 gene of the lager brewing yeast (cer-BAP2) is very similar to the Saccharomyces cerevisiae BAP2 gene. This result substantiates the notion that lager brewing yeast is a hybrid of S. cerevisiae and S. bayanus. The amino acid sequence homology between S. cerevisiae Bap2p and Lg-Bap2p was 88%. The transcription of Lg-BAP2 was not induced by the addition of leucine to the growth medium, while that of cer-BAP2 was induced. The transcription of Lg-BAP2 was repressed by the presence of ethanol and weak organic acid, while that of cer-BAP2 was not affected by these compounds. Furthermore, Northern analysis during beer fermentation revealed that the transcription of Lg-BAP2 was repressed at the beginning of the fermentation, while cer-BAP2 was highly expressed throughout the fermentation. These results suggest that the transcription of Lg-BAP2 is regulated differently from that of cer-BAP2 in lager brewing yeasts.

The branched-chain amino acid permease Bap2p is a transport system for leucine, isoleucine, and valine in Saccharomyces cerevisiae, and its synthesis is regulated transcriptionally. However, the downregulation mechanisms of Bap2p have not been established. Here we demonstrate that the C-terminal region of Bap2p plays a pivotal role in its basal turnover. Truncation of the C-terminal 29 residues caused the stabilization and accumulation in the plasma membrane of Bap2p. Furthermore, when the Bap2p C-terminal region was fused to green fluorescent protein, the fusion protein localized to the plasma membrane, suggesting the existence of a possible degradation-related acceptor site for the C-terminal tail of Bap2p.

Transcription of MET genes in Saccharomyces cerevisiae depends on a transcriptional activator, the MET4 gene product (Met4p). Using in vitro mutagenesis, we isolated two mutant MET4 alleles encoding [Pro215]Met4p and [Ser156]Met4p. These mutations impeded Met4p's responsiveness to methionine in the media, and yeast cells carrying mutant alleles exhibited enhanced transcription of MET genes under repressing conditions. The enhanced transcription was dependent on the CBF1 gene, but did not compete with an excess of wild-type Met4p, suggesting that some changes in the affinity of Met4p to other factors might be involved in S-adenosylmethionine-mediated transcriptional regulation.