Variation in 4-mercapto-4-methyl-pentan-2-one release by Saccharomyces cerevisiae commercial wine strains

The volatile thiol 4-mercapto-4-methylpentan-2-one (4MMP) is a potent contributor to wine aroma. In grape juice, 4MMP is bound to cysteine as a non-volatile compound and requires the action of yeast during fermentation to release the aroma active thiol. A method was developed to measure 4MMP release from the precursor by headspace solid-phase microextraction and separation by gas chromatography with atomic emission detection to screen the ability of wine yeast to release 4MMP. Yeast commonly used in white wine making were grown with the precursor at two different temperatures, and the amount of 4MMP released was measured. The results demonstrate that yeast strain selection and fermentation temperature can provide an important tool to enhance or modulate the grape-derived aromas formed during wine fermentation.     

Molecular study on the role of vacuolar transporters in glycyrrhetinic acid production in engineered Saccharomyces cerevisiae

Glycyrrhetinic acid (GA) is one of the major bioactive components of the leguminous plant, Glycyrrhiza spp. (Chinese licorice). Owing to GA's complicated chemical structure, its production by chemical synthesis is challenging and requires other efficient strategies such as microbial synthesis. Earlier investigations employed numerous approaches to improve GA yield by refining the synthetic pathway and improving the metabolic flux. Nevertheless, the metabolic role of transporters in GA biosynthesis in microbial cell factories has not been studied so far. In this study, we investigated the role of yeast ATP binding cassette (ABC) vacuolar transporters in GA production. Molecular docking of GA and its precursors, β-Amyrin and 11-oxo-β-amyrin, was performed with five vacuolar ABC transporters (Bpt1p, Vmr1p, Ybt1p, Ycf1p and Nft1p). Based on docking scores, two top scoring transporters were selected (Bpt1p and Vmr1p) to investigate transporters' functions on GA production via overexpression and knockout experiments in one GA-producing yeast strain (GA166). Results revealed that GA and its precursors exhibited the highest predicted binding affinity towards BPT1 (ΔG = ?10.9, ?10.6, ?10.9 kcal/mol for GA, β-amyrin and 11-oxo-β-amyrin, respectively). Experimental results showed that the overexpression of BPT1 and VMR1 restored the intracellular as well as extracellular GA production level under limited nutritional conditions, whereas knockout of BPT1 resulted in a total loss of GA production. These results suggest that the activity of BPT1 is required for GA production in engineered Saccharomyces cerevisiae.     

Mutational analysis of Vam4/Ypt7p function in the vacuolar biogenesis and morphogenesis in the yeast,Saccharomyces cerevisiae

Summary The vacuole is one of the most prominent compartments in yeast cells. The wild-type yeast cells have a large vacuolar compartment which occupies approximately a quarter of the cell volume, while thevam4 mutant cells exhibit highly fragmented vacuolar morphology. We isolated theVAM4 gene and found that theVAM4 is identical to theYPT7 which encodes a member of small GTP-binding protein superfamily. We introduced mutations to theVAM4/YPT7 which alter nucleotide binding characteristics of the gene product specifically, and their activities for the vacuolar morphogenesis were examined by transforming the mutant genes into yeast cells. The Thr22Asn mutation, which was expected to fix the protein in the GDP-bound state, resulted in loss of function in the vacuolar morphogenesis. Subcellular fractionation analysis indicated that the mutant molecule did not associate with intracellular membranes efficiently. In contrast, Vam4/Ypt7p with the Gln68Leu mutation, which was expected to be the GTP-bound form, complemented the fragmented vacuolar morphology of vam4 mutant cells. Vam4/Ypt7p with the Gln68Leu mutation also complemented the defects in the biogenesis of vacuolar alkaline phosphatase whose maturation requires the proper function of Vam4/Ypt7p. Overexpression of the mutant proteins in wild-type cells did not develop dominant-negative effects on the vacuolar assembly. These results indicated that the GTP-bound form of Vam4/Ypt7p promotes the biogenesis and morphogenesis of the yeast vacuolar compartment.Abbreviations ALP alkaline phosphatase - CDE centromeric - DNA element - CPY carboxypeptidase Y - GST glutathione S-transferase     

Structure and function of the GTP binding protein Gtr1 and its role in phosphate transport in Saccharomyces cerevisiae

The Pho84 high-affinity phosphate permease is the primary phosphate transporter in the yeast Saccharomyces cerevisiae under phosphate-limiting conditions. The soluble G protein, Gtr1, has previously been suggested to be involved in the derepressible Pho84 phosphate uptake function. This idea was based on a displayed deletion phenotype of Deltagtr1 similar to the Deltapho84 phenotype. As of yet, the mode of interaction has not been described. The consequences of a deletion of gtr1 on in vivo Pho84 expression, trafficking and activity, and extracellular phosphatase activity were analyzed in strains synthesizing either Pho84-green fluorescent protein or Pho84-myc chimeras. The studies revealed a delayed response in Pho84-mediated phosphate uptake and extracellular phosphatase activity under phosphate-limiting conditions. EPR spectroscopic studies verified that the N-terminal G binding domain (residues 1-185) harbors the nucleotide responsive elements. In contrast, the spectra obtained for the C-terminal part (residues 186-310) displayed no evidence of conformational changes upon GTP addition.     

The nuclear GTPase Gsp1p can affect proper telomeric function through the Sir4 protein in Saccharomyces cerevisiae

The small Ras-like GTPase Ran/Gsp1p is a highly conserved nuclear protein required for the nucleocytoplasmic trafficking of macromolecules. Recent findings suggest that the Ran/Gsp1p pathway may have additional roles in several aspects of nuclear structure and function, including spindle assembly, nuclear envelope formation, nuclear pore complex assembly and RNA processing. Here, we provide evidence that Gsp1p can regulate telomeric function in Saccharomyces cerevisiae. We show that overexpression of PRP20, encoding the Gsp1p GDP/GTP nuclear exchange factor, specifically weakens telomeric silencing without detectably affecting nucleocytoplasmic transport. In addition to this silencing defect, we show that Rap1p and Sir3p delocalize from their normal telomeric foci. Interestingly, Gsp1p was found to interact genetically and physically with the telomeric component Sir4p. Taken together, these results suggest that the GSP1 pathway could regulate proper telomeric function in yeast through Sir4p.     

GLR1 plays an essential role in the homeodynamics of glutathione and the regulation of H2S production during respiratory oscillation of Saccharomyces cerevisiae

The role of glutathione (GSH) and its homeodynamics during respiratory oscillation of Saccharomyces cerevisiae were investigated. Pulse injection of thiol redox modifying agents, such as diethylmaleate, N-ethylmaleimide, DL-butione-[S,R]-sulfoxamine, or 5-nitro-2-furaldehyde into the culture perturbed oscillation, although the degree of perturbation varied. Analysis of the expression profiles of GSH1 and GLR1, the activities of glutathione reductase, oscillations in cysteine and GSH concentrations, and the chemostat culture of the GLR1 disruptant indicated that GLR1 plays an essential role in the homeodynamics of GSH and the regulation of H(2)S production.     

The role of the Brr5/Ysh1 C-terminal domain and its homolog Syc1 in mRNA 3'-end processing in Saccharomyces cerevisiae

The cleavage/polyadenylation factor (CPF) of Saccharomyces cerevisiae is thought to provide the catalytic activities of the mRNA 3'-end processing machinery, which include endonucleolytic cleavage at the poly(A) site, followed by synthesis of an adenosine polymer onto the new 3'-end by the CPF subunit Pap1. Because of similarity to other nucleases in the metallo-beta-lactamase family, the Brr5/Ysh1 subunit has been proposed to be the endonuclease. The C-terminal domain of Brr5 lies outside of beta-lactamase homology, and its function has not been elucidated. We show here that this region of Brr5 is necessary for cell viability and mRNA 3'-end processing. It is highly homologous to another CPF subunit, Syc1. Syc1 is not essential, but its removal improves the growth of other processing mutants at restrictive temperatures and restores in vitro processing activity to cleavage/ polyadenylation-defective brr5-1 extract. Our findings suggest that Syc1, by mimicking the essential Brr5 C-terminus, serves as a negative regulator of mRNA 3'-end formation.     

The Saccharomyces cerevisiae actin cytoskeletal component Bsp1p has an auxiliary role in actomyosin ring function and in the maintenance of bud-neck structure

Iqg1p is a component of the actomyosin contractile ring that is required for actin recruitment and septum deposition. Cells lacking Iqg1p function have an altered bud-neck structure and fail to form a functional actomyosin contractile ring resulting in a block to cytokinesis and septation. Here it is demonstrated that increased expression of the actin cytoskeleton associated protein Bsp1p bypasses the requirement for contractile ring function. This also correlates with reduced bud-neck width and remedial septum formation. Increased expression of this protein in a temperature-sensitive iqg1-1 background causes remedial septum formation at the bud neck that is reliant upon chitin synthase III activity and restores cell separation. The observed suppression correlates with a restoration of normal bud-neck structure. While Bsp1p is a component of the contractile ring, its recruitment to the bud neck is not required for the observed suppression. Loss of Bsp1p causes a brief delay in the redistribution of the actin cytoskeleton normally observed at the end of actin ring contraction. Compromise of Iqg1p function, in the absence of Bsp1p function, leads to a profound change in the distribution of actin and the pattern of cell growth accompanied by a failure to complete cytokinesis and cell separation.     

Genetic analysis of the Saccharomyces cerevisiae Sgs1 helicase defines an essential function for the Sgs1-Top3 complex in the absence of SRS2 or TOP1

The Saccharomyces cerevisiae gene SGS1 encodes a DNA helicase that shows homology to the Escherichia coli protein RecQ and the products of the BLM and WRN genes in humans, which are defective in Bloom's and Werner's syndrome, respectively. Recently, it has been proposed that this helicase is involved in maintaining the integrity of the rDNA and that loss of Sgs1 function leads to accelerated aging. Sgs1 has been isolated on the basis of its genetic interaction with both topoisomerase I and topoisomerase III, as well as in a two-hybrid screen for proteins that interact with the C-terminal portion of topoisomerase II. We have defined the minimal structural elements of Sgs1 required for its interactions with the three topoisomerases, and demonstrate that the complex phenotypes associated with sgs1 mutants are a consequence of a dysfunctional Sgs1-Top3 complex. We also report that the synthetic relationship between mutations in SGS1 and SRS2, which encodes another helicase implicated in recombinational repair, likewise result from a dysfunctional Sgs1-Top3 interaction. Our findings indicate that Sgs1 may act on different DNA structures depending on the activity of topoisomerase I, Srs2 and topoisomerase III.     

Evidence for the genetic interaction between the actin-binding protein Vrp1 and the RhoGAP Rgd1 mediated through Rho3p and Rho4p in Saccharomyces cerevisiae

The non-essential RGD1 gene from Saccharomyces cerevisiae encodes a protein that has been characterized in vitro as a Rho GTPase activating protein (RhoGAP) for the Rho3 and Rho4 proteins. Rgd1p, which displays a conserved FCH-coiled coil-Rho-GAP domain organization, showed a patch-like distribution in the cell, including a localization in growing buds. Using a genetic screen, we found that rgd1delta and vrp1alpha mutations exhibited a synthetic lethality, thus revealing an interaction between these genes. The VRP1 product is an actin and myosin interacting protein involved in polarized growth. Using mutant forms of both Rho3 and Rho4 proteins, we provide evidence for the involvement of these two GTPases in RGD1-VRP1 co-lethality. In addition, these results strongly argue in favour of Rho3p and Rho4p being the targets of Rgd1p RhoGAP activity in vivo. Genetic relationships between either VRP1 or RGD1 and actin cytoskeleton-linked genes were also studied. These and other well-established data support the idea that Vrp1, Las17, Rvs167 proteins belong to the same complex. This protein structure might act with myosins in various actin cytoskeleton-based activities, in co-operation with a Rho3p/Rho4p signalling pathway that is negatively regulated by Rgd1p GAP activity.     

Genetic study of interactions between the cytoskeletal assembly protein sla1 and prion-forming domain of the release factor Sup35 (eRF3) in Saccharomyces cerevisiae.

Striking similarities between cytoskeletal assembly and the "nucleated polymerization" model of prion propagation suggest that similar or overlapping sets of proteins may assist in both processes. We show that the C-terminal domain of the yeast cytoskeletal assembly protein Sla1 (Sla1C) specifically interacts with the N-terminal prion-forming domain (Sup35N) of the yeast release factor Sup35 (eRF3) in the two-hybrid system. Sla1C and several other Sup35N-interacting proteins also exhibit two-hybrid interactions with the poly-Gln-expanded N-proximal fragment of human huntingtin, which promotes Huntington disease-associated aggregation. The Sup35N-Sla1C interaction is inhibited by Sup35N alterations that make Sup35 unable to propagate the [PSI(+)] state and by the absence of the chaperone protein Hsp104, which is essential for [PSI] propagation. In a Sla1(-) background, [PSI] curing by dimethylsulfoxide or excess Hsp104 is increased, while translational readthrough and de novo [PSI] formation induced by excess Sup35 or Sup35N are decreased. These data show that, in agreement with the proposed function of Sla1 during cytoskeletal formation, Sla1 assists in [PSI] formation and propagation, but is not required for these processes. Sla1(-) strains are sensitive to some translational inhibitors, and some sup35 mutants, obtained in a Sla1(-) background, are sensitive to Sla1, suggesting that the interaction between Sla1 and Sup35 proteins may play a role in the normal function of the translational apparatus. We hypothesize that Sup35N is involved in regulatory interactions with intracellular structural networks, and [PSI] prion may be formed as a by-product of this process.     

Interaction between the poly(A)-binding protein Pab1 and the eukaryotic release factor eRF3 regulates translation termination but not mRNA decay in Saccharomyces cerevisiae

Eukaryotic release factor 3 (eRF3) is implicated in translation termination and also interacts with the poly(A)-binding protein (PABP, Pab1 in yeast), a major player in mRNA metabolism. Despite conservation of this interaction, its precise function remains elusive. First, we showed experimentally that yeast eRF3 does not contain any obvious consensus PAM2 (PABP-interacting motif 2). Thus, in yeast this association is different from the well described interaction between the metazoan factors. To gain insight into the exact function of this interaction, we then analyzed the phenotypes resulting from deleting the respective binding domains. Deletion of the Pab1 interaction domain on eRF3 did not affect general mRNA stability or nonsense-mediated mRNA decay (NMD) pathway and induced a decrease in translational readthrough. Furthermore, combined deletions of the respective interacting domains on eRF3 and on Pab1 were viable, did not affect Pab1 function in mRNA stability and harbored an antisuppression phenotype. Our results show that in Saccharomyces cerevisiae the role of the Pab1 C-terminal domain in mRNA stability is independent of eRF3 and the association of these two factors negatively regulates translation termination.     

A RING-H2 finger motif is essential for the function of Der3/Hrd1 in endoplasmic reticulum associated protein degradation in the yeast Saccharomyces cerevisiae

Der3/Hrd1p is a protein required for proper degradation of misfolded soluble and integral membrane proteins in the endoplasmic reticulum (ER) in the yeast Saccharomyces cerevisiae. It is located to the ER membrane and consists of a N-terminal hydrophobic region with several transmembrane domains and a large hydrophilic tail oriented to the ER lumen containing a RING finger motif of the H2 class. We had previously reported that a truncated version of Der3p, Der3deltaRp, lacking 111 residues of the lumenal domain including the RING finger motif is not functional, suggesting the involvement of this domain in the function of the protein in ER degradation. We substantiated this hypothesis by constructing a mutated form of Der3/Hrd1p replacing the last cysteine of the motif with a serine. This mutated Der3(C399S) protein maintains the correct localization and topology of the wild-type protein, however, is not able to support the degradation of soluble and integral membrane proteins. This point mutation altering the RING-H2 motif behaves as a dominant allele especially when overexpressed from a 2mu plasmid by this increasing the half-life of CPY* more than 6-fold when compared with a wild-type strain. Furthermore coexpression of der3(C399S) with the wild-type allele is also able to partially suppress the temperature sensitive growth phenotype of a sec61-2 strain. Finally we have shown that overexpression of Hrd3p suppresses the dominant effect of the der3(C399S) mutation. These results could be explained by a competition between wild-type and mutant Der3 protein for the interaction with some other component of the ER degradation pathway, probably Hrd3p.