Dynamics of the yeast transcriptome during wine fermentation reveals a novel fermentation stress response

In this study, genome-wide expression analyses were used to study the response of Saccharomyces cerevisiae to stress throughout a 15-day wine fermentation. Forty per cent of the yeast genome significantly changed expression levels to mediate long-term adaptation to fermenting grape must. Among the genes that changed expression levels, a group of 223 genes was identified, which was designated as fermentation stress response (FSR) genes that were dramatically induced at various points during fermentation. FSR genes sustain high levels of induction up to the final time point and exhibited changes in expression levels ranging from four- to 80-fold. The FSR is novel; 62% of the genes involved have not been implicated in global stress responses and 28% of the FSR genes have no functional annotation. Genes involved in respiratory metabolism and gluconeogenesis were expressed during fermentation despite the presence of high concentrations of glucose. Ethanol, rather than nutrient depletion, seems to be responsible for entry of yeast cells into the stationary phase.     

Assignment of the human glycogen debrancher gene to chromosome 1p21

Glycogen debranching enzyme is a monomeric protein containing two independent catalytic activities of glycantransferase and glucosidase that are both required for glycogen degradation. Its deficiency causes type III glycogen storage disease. A majority of the patients with this disease have deficient enzyme activity in both liver and muscle (type IIIa) but approximately 15% of them lack enzyme activity only in the liver (type IIIb); however, the enzyme is a monomer and appears to be identical in all the tissues. The cDNA coding for the complete human muscle debranching enzyme has recently been isolated. Using the cDNA clones, the debrancher gene was localized to human chromosome 1 by somatic cell hybrid analysis. Regional assignment to chromosome band 1p21 was determined by in situ hybridization. Mapping of the debrancher gene to a single chromosome site is consistent with our hypotheses that a single gene encodes both liver and muscle debrancher protein.     

Determination of yeast glycogen content by individual cell spectroscopy using image analysis

A rapid technique has been developed to determine the glycogen content of yeast on an individual cell basis using a combination of image analysis technology and staining of yeast cells with an I(2):KI solution. Changes in mean cellular glycogen content during alcoholic fermentation have been reported using this technique. The glycogen content of stored brewer's yeast is heterogeneous compared to freshly propagated yeast which have a more uniform distribution of glycogen. Analysis of the distribution of yeast glycogen during fermentation indicates that a fraction of yeast cells do not dissimilate glycogen. Therefore, conventional analysis of the mean glycogen content of yeast used to inoculate fermentations is of limited use, unless information regarding the proportion of cells which utilize glycogen is known. Analysis of the distribution of glycogen within a yeast population can serve as a useful indicator of yeast quality.     

Antagonistic controls of autophagy and glycogen accumulation by Snf1p, the yeast homolog of AMP-activated protein kinase, and the cyclin-dependent kinase Pho85p

In the yeast Saccharomyces cerevisiae, glycogen is accumulated as a carbohydrate reserve when cells are deprived of nutrients. Yeast mutated in SNF1, a gene encoding a protein kinase required for glucose derepression, has diminished glycogen accumulation and concomitant inactivation of glycogen synthase. Restoration of synthesis in an snf1 strain results only in transient glycogen accumulation, implying the existence of other SNF1-dependent controls of glycogen storage. A genetic screen revealed that two genes involved in autophagy, APG1 and APG13, may be regulated by SNF1. Increased autophagic activity was observed in wild-type cells entering the stationary phase, but this induction was impaired in an snf1 strain. Mutants defective for autophagy were able to synthesize glycogen upon approaching the stationary phase, but were unable to maintain their glycogen stores, because subsequent synthesis was impaired and degradation by phosphorylase, Gph1p, was enhanced. Thus, deletion of GPH1 partially reversed the loss of glycogen accumulation in autophagy mutants. Loss of the vacuolar glucosidase, SGA1, also protected glycogen stores, but only very late in the stationary phase. Gph1p and Sga1p may therefore degrade physically distinct pools of glycogen. Pho85p is a cyclin-dependent protein kinase that antagonizes SNF1 control of glycogen synthesis. Induction of autophagy in pho85 mutants entering the stationary phase was exaggerated compared to the level in wild-type cells, but was blocked in apg1 pho85 mutants. We propose that Snf1p and Pho85p are, respectively, positive and negative regulators of autophagy, probably via Apg1 and/or Apg13. Defective glycogen storage in snf1 cells can be attributed to both defective synthesis upon entry into stationary phase and impaired maintenance of glycogen levels caused by the lack of autophagy.     

Analysis of Saccharomyces cerevisiae hexose carrier expression during wine fermentation: both low- and high-affinity Hxt transporters are expressed

The transport of glucose and fructose into yeast cells is a critical step in the utilization of sugars during wine fermentation. Hexose uptake can be carried out by various Hxt carriers, each possessing distinct regulatory and transport-kinetic properties capable of influencing yeast fermentation capacity. We investigated the expression pattern of the hexose transporters Hxt1 to 7 at the promoter and protein levels in Saccharomyces cerevisiae during wine fermentation. The Hxt1p carrier was expressed only at the beginning of fermentation, and had no role during stationary phase. The Hxt3p carrier was the only one to be expressed throughout fermentation, displaying maximal expression at growth arrest and slowly decreasing in abundance over the course of the stationary phase. The high-affinity carriers Hxt6p and Hxt7p displayed similar expression profiles, with expression induced at entry into stationary phase and persisting throughout the phase. The expression of these two carriers occurred despite the presence of high amounts of hexoses, and the proteins were stably expressed when the cells were starved for nitrogen. The Hxt2p transporter was only transiently expressed during lag phase, which suggests a role for the protein in growth initiation. Characterization of glucose transport kinetics indicated the presence of a shift in the low-affinity component that is consistent with a predominant expression of Hxt1p during growth phase and of Hxt3p during stationary phase. In addition, a high-affinity uptake component consistent with functional expression of Hxt6p/Hxt7p was identified during stationary phase.     

Regulation of yeast glycogen metabolism and sporulation by Glc7p protein phosphatase.

Glc7p is an essential serine/threonine type 1 protein phosphatase (PP1) from the yeast Saccharomyces cerevisiae, which has a role in many processes including cell cycle progression, sporulation, glycogen accumulation, translation initiation, and glucose repression. Two hallmarks of PP1 enzymes are very high amino acid sequence conservation and association of the catalytic subunit with a variety of noncatalytic, regulatory subunits. We tested the hypothesis that PP1 sequence conservation was the result of each PP1 residue playing a role in multiple intermolecular interactions. Analysis of 24 glc7 mutants, isolated primarily by their glycogen accumulation traits, revealed that every mutated Glc7p residue altered many noncatalytic subunit affinities and conferred unselected sporulation traits to various degrees. Furthermore, quantitative analysis showed that Glc7p affinity for the glycogen-binding noncatalytic subunit Gac1p was not the only parameter that determines the glycogen accumulation by a glc7 mutant. Sds22p is one Glc7p noncatalytic subunit that is essential for mitotic growth. Surprisingly, several mutant Glc7p proteins had undetectable affinity for Sds22p, yet grew apparently normally. The characterization of glc7 diploid sporulation revealed that Glc7p has at least two meiotic roles. Premeiotic DNA synthesis was undetectable in glc7 mutants with the poorest sporulation. In the glc7 diploids examined, expression of the meiotic inducer IME1 was proportional to the glc7 diploid sporulation frequency. Moreover, IME1 hyperexpression could not suppress glc7 sporulation traits. The Glc7p/Gip1p holoenzyme may participate in completion of meiotic divisions or spore packaging because meiotic dyads predominate when some glc7 diploids sporulate.     

The Saccharomyces cerevisiae YFR041C/ERJ5 gene encoding a type I membrane protein with a J domain is required to preserve the folding capacity of the endoplasmic reticulum

YFR041C/ERJ5 was identified in Saccharomyces cerevisiae as a gene regulated by the unfolded protein response pathway (UPR). The open reading frame of the gene has a J domain characteristic of the DnaJ chaperone family of proteins that regulate the activity of Hsp70 chaperones. We determined the expression and topology of Erj5p, a type I membrane protein with a J domain in the lumen of the endoplasmic reticulum (ER) that colocalizes with Kar2p, the major Hsp70 in the yeast ER. We identified synthetic interactions of Deltaerj5 with mutations in genes involved in protein folding in the ER (kar2-159, Deltascj1Deltajem1) and in the induction of the unfolded protein response (Deltaire1). Loss of Erj5p in yeast cells with impaired ER protein folding capacity increased sensitivity to agents that cause ER stress. We identified the ERJ5 mRNA and confirmed that agents that promote accumulation of misfolded proteins in the ER regulate its abundance. We found that loss of the non-essential ERJ5 gene leads to a constitutively induced UPR, indicating that ERJ5 is required for maintenance of an optimal folding environment in the yeast ER.     

Requirement of the self-glucosylating initiator proteins Glg1p and Glg2p for glycogen accumulation in Saccharomyces cerevisiae.

Glycogen, a branched polymer of glucose, is a storage molecule whose accumulation is under rigorous nutritional control in many cells. We report the identification of two Saccharomyces cerevisiae genes, GLG1 and GLG2, whose products are implicated in the biogenesis of glycogen. These genes encode self-glucosylating proteins that in vitro can act as primers for the elongation reaction catalyzed by glycogen synthase. Over a region of 258 residues, the Glg proteins have 55% sequence identify to each other and approximately 33% identity to glycogenin, a mammalian protein postulated to have a role in the initiation of glycogen biosynthesis. Yeast cells defective in either GLG1 or GLG2 are similar to the wild type in their ability to accumulate glycogen. Disruption of both genes results in the inability of the cells to synthesize glycogen despite normal levels of glycogen synthase. These results suggest that a self-glucosylating protein is required for glycogen biosynthesis in a eukaryotic cell. The activation state of glycogen synthase in glg1 glg2 cells is suppressed, suggesting that the Glg proteins may additionally influence the phosphorylation state of glycogen synthase.     

Pho85p, a cyclin-dependent protein kinase, and the Snf1p protein kinase act antagonistically to control glycogen accumulation in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, nutrient levels control multiple cellular processes. Cells lacking the SNF1 gene cannot express glucose-repressible genes and do not accumulate the storage polysaccharide glycogen. The impaired glycogen synthesis is due to maintenance of glycogen synthase in a hyperphosphorylated, inactive state. In a screen for second site suppressors of the glycogen storage defect of snf1 cells, we identified a mutant gene that restored glycogen accumulation and which was allelic with PHO85, which encodes a member of the cyclin-dependent kinase family. In cells with disrupted PHO85 genes, we observed hyperaccumulation of glycogen, activation of glycogen synthase, and impaired glycogen synthase kinase activity. In snf1 cells, glycogen synthase kinase activity was elevated. Partial purification of glycogen synthase kinase activity from yeast extracts resulted in the separation of two fractions by phenyl-Sepharose chromatography, both of which phosphorylated and inactivated glycogen synthase. The activity of one of these, GPK2, was inhibited by olomoucine, which potently inhibits cyclin-dependent protein kinases, and contained an approximately 36-kDa species that reacted with antibodies to Pho85p. Analysis of Ser-to-Ala mutations at the three potential Gsy2p phosphorylation sites in pho85 cells implicated Ser-654 and/or Thr-667 in PHO85 control of glycogen synthase. We propose that Pho85p is a physiological glycogen synthase kinase, possibly acting downstream of Snf1p.     

The yeast cyclins Pc16p and Pc17p are involved in the control of glycogen storage by the cyclin-dependent protein kinase Pho85p

Pho85p is a yeast cyclin-dependent protein kinase (Cdk) that can interact with 10 cyclins (Pcls) to form multiple protein kinases. The functions of most of the Pcls, including Pc16p and Pc17p, are poorly defined. We report here that Pc16p and Pc17p are involved in the metabolism of the branched storage polysaccharide glycogen under certain conditions and deletion of PCL6 and PCL7 restores glycogen accumulation to a snf1 pcl8 pcl10 triple mutant, paradoxically activating both glycogen synthase and phosphorylase. Pho85p thus affects glycogen accumulation through multiple Cdks composed of different cyclin partners.     

Roles of trehalose phosphate synthase in yeast glycogen metabolism and sporulation

Trehalose is a major storage carbohydrate in budding yeast, Saccharomyces cerevisiae. Alterations in trehalose synthesis affect carbon source-dependent growth, accumulation of glycogen and sporulation. Trehalose is synthesized by trehalose phosphate synthase (TPS), which is a complex of at least four proteins. In this work, we show that the Tps1p subunit protein catalyses trehalose phosphate synthesis in the absence of other TPS components. The tps1-H223Y allele (glc6-1) that causes a semidominant decrease in glycogen accumulation exhibits greater enzyme activity than wild-type TPS1 because, unlike the wild-type enzyme, TPS activity in tps1-H223Y cells is not inhibited by phosphate. Poor sporulation in tps1 null diploids is caused by reduced expression of meiotic inducers encoded by IME1, IME2 and MCK1. Furthermore, high-copy MCK1 or heterozygous hxk2 mutations can suppress the tps1 sporulation trait. These results suggest that the trehalose-6-phosphate inhibition of hexokinase activity is required for full induction of MCK1 in sporulating yeast cells.     

Partitioning-defective protein 6 regulates insulin-dependent glycogen synthesis via atypical protein kinase C

The atypical isoforms of protein kinase C (aPKCs) play an important role in insulin signaling and are involved in insulin-stimulated glucose uptake in different cell systems. On the other hand, aPKCs also are able to negatively regulate important proteins for insulin signaling, like phosphatidylinositol 3-kinase and protein kinase B/Akt. To find aPKC-interacting proteins that may promote positive or negative activities of aPKCs, a yeast two-hybrid screen was performed. Partitioning-defective protein 6 (Par6) was detected in human cDNA libraries of different adult insulin-sensitive tissues. Although Par6 is known as an aPKC-interacting protein during development, no role for Par6 in insulin signaling has been reported so far. We therefore studied the effects of Par6 overexpression in C2C12 murine myoblasts. In these cells, Par6 associated constitutively with endogenous aPKCs, and the expression level as well as the activity of aPKCs were increased. Insulin-dependent association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate 1 was hampered and the phosphorylation of Akt/glycogen synthase kinase-3alpha/beta was significantly impaired after stimulation with insulin or with platelet-derived growth factor. Consequently, insulin-dependent glycogen synthesis was down-regulated (1.44 vs. 2.24 fold, P < 0.01). We therefore suggest that Par6 acts as a negative regulator of the insulin signal.     

Multiple glycogen-binding sites in eukaryotic glycogen synthase are required for high catalytic efficiency toward glycogen

Glycogen synthase is a rate-limiting enzyme in the biosynthesis of glycogen and has an essential role in glucose homeostasis. The three-dimensional structures of yeast glycogen synthase (Gsy2p) complexed with maltooctaose identified four conserved maltodextrin-binding sites distributed across the surface of the enzyme. Site-1 is positioned on the N-terminal domain, site-2 and site-3 are present on the C-terminal domain, and site-4 is located in an interdomain cleft adjacent to the active site. Mutation of these surface sites decreased glycogen binding and catalytic efficiency toward glycogen. Mutations within site-1 and site-2 reduced the V(max)/S(0.5) for glycogen by 40- and 70-fold, respectively. Combined mutation of site-1 and site-2 decreased the V(max)/S(0.5) for glycogen by >3000-fold. Consistent with the in vitro data, glycogen accumulation in glycogen synthase-deficient yeast cells (Δgsy1-gsy2) transformed with the site-1, site-2, combined site-1/site-2, or site-4 mutant form of Gsy2p was decreased by up to 40-fold. In contrast to the glycogen results, the ability to utilize maltooctaose as an in vitro substrate was unaffected in the site-2 mutant, moderately affected in the site-1 mutant, and almost completely abolished in the site-4 mutant. These data show that the ability to utilize maltooctaose as a substrate can be independent of the ability to utilize glycogen. Our data support the hypothesis that site-1 and site-2 provide a "toehold mechanism," keeping glycogen synthase tightly associated with the glycogen particle, whereas site-4 is more closely associated with positioning of the nonreducing end during catalysis.     

A loss-of-function mutation in the PAS kinase Rim15p is related to defective quiescence entry and high fermentation rates of Saccharomyces cerevisiae sake yeast strains

Sake yeast cells have defective entry into the quiescent state, allowing them to sustain high fermentation rates. To reveal the underlying mechanism, we investigated the PAS kinase Rim15p, which orchestrates initiation of the quiescence program in Saccharomyces cerevisiae. We found that Rim15p is truncated at the carboxyl terminus in modern sake yeast strains as a result of a frameshift mutation. Introduction of this mutation or deletion of the full-length RIM15 gene in a laboratory strain led to a defective stress response, decreased synthesis of the storage carbohydrates trehalose and glycogen, and impaired G(1) arrest, which together closely resemble the characteristic phenotypes of sake yeast. Notably, expression of a functional RIM15 gene in a modern sake strain suppressed all of these phenotypes, demonstrating that dysfunction of Rim15p prevents sake yeast cells from entering quiescence. Moreover, loss of Rim15p or its downstream targets Igo1p and Igo2p remarkably improved the fermentation rate in a laboratory strain. This finding verified that Rim15p-mediated entry into quiescence plays pivotal roles in the inhibition of ethanol fermentation. Taken together, our results suggest that the loss-of-function mutation in the RIM15 gene may be the key genetic determinant of the increased ethanol production rates in modern sake yeast strains.     

Hyperactive glycogen synthase mutants of Saccharomyces cerevisiae suppress the glc7-1 protein phosphatase mutant

A yeast glc7-1 mutant expressing a variant of protein phosphatase type 1 fails to accumulate glycogen. This defect is associated with hyperphosphorylated and inactive glycogen synthase, consistent with Glc7p acting directly to dephosphorylate and activate glycogen synthase. To characterize the glycogen synthesis defect of this mutant in more detail, we isolated 26 pseudorevertants of the glc7-1 mutant. All pseudoreversion events were due to missense mutations in GSY2, the gene encoding the major isoform of glycogen synthase. A majority of the mutations responsible for the suppression were in the 3' end of the gene, corresponding to the phosphorylated COOH terminus of Gsy2p. Phosphorylation of the mutant proteins was reduced, suggesting that they are poor substrates for glycogen synthase kinases. Suppressor mutations outside this domain did not decrease the phosphorylation of the resulting proteins, indicating that these proteins are immune to the regulatory effects of phosphorylation. Since no growth defect has been observed for strains with altered glycogen levels, the relative levels of fitness of GSY2 mutants that fail to accumulate glycogen and that hyperaccumulate glycogen were assayed by cocultivation experiments. A wild-type strain outcompeted both hypo- and hyperaccumulating strains, suggesting that glycogen levels contribute substantially to the fitness of yeast.     

Yeast Ran-binding protein Yrb1p is required for efficient proteolysis of cell cycle regulatory proteins Pds1p and Sic1p

Ubiquitin-dependent proteolysis of specific target proteins is required for several important steps during the cell cycle. Degradation of such proteins is strictly cell cycle-regulated and triggered by two large ubiquitin ligases, termed anaphase-promoting complex (APC) and Skp1/Cullin/F-box complex (SCF). Here we show that yeast Ran-binding protein 1 (Yrb1p), a predominantly cytoplasmic protein implicated in nucleocytoplasmic transport, is required for cell cycle regulated protein degradation. Depletion of Yrb1p results in the accumulation of unbudded G(1) cells and of cells arrested in mitosis implying a function of Yrb1p in the G(1)/S transition and in the progression through mitosis. Temperature-sensitive yrb1-51 mutants are defective in APC-mediated degradation of the anaphase inhibitor protein Pds1p and in degradation of the cyclin-dependent kinase inhibitor Sic1p, a target of SCF. Thus, Yrb1p is crucial for efficient APC- and SCF-mediated proteolysis of important cell cycle regulatory proteins. We have identified the UBS1 gene as a multicopy suppressor of yrb1-51 mutants. Ubs1p is a nuclear protein, and its deletion is synthetic lethal with a yrb1-51 mutation. Interestingly, UBS1 was previously identified as a multicopy suppressor of cdc34-2 mutants, which are defective in SCF activity. We suggest that Ubs1p may represent a link between nucleocytoplasmic transport and ubiquitin ligase activity.     

Role of the Escherichia coli glgX gene in glycogen metabolism

A role for the Escherichia coli glgX gene in bacterial glycogen synthesis and/or degradation has been inferred from the sequence homology between the glgX gene and the genes encoding isoamylase-type debranching enzymes; however, experimental evidence or definition of the role of the gene has been lacking. Construction of E. coli strains with defined deletions in the glgX gene is reported here. The results show that the GlgX gene encodes an isoamylase-type debranching enzyme with high specificity for hydrolysis of chains consisting of three or four glucose residues. This specificity ensures that GlgX does not generate an extensive futile cycle during glycogen synthesis in which chains with more than four glucose residues are transferred by the branching enzyme. Disruption of glgX leads to overproduction of glycogen containing short external chains. These results suggest that the GlgX protein is predominantly involved in glycogen catabolism by selectively debranching the polysaccharide outer chains that were previously recessed by glycogen phosphorylase.