Solution phase synthesis of Saccharomyces cerevisiae a-mating factor and its analogs

The solution phase synthesis of the Saccharomyces cerevisiae a-mating factor and nonfarnesylated and nonmethylated a-factor analogs are reported. The a-factor, a lipopeptide with the sequence Tyr-Ile-Ile-Lys-Gly-Val-Phe-Trp-Asp-Pro-Ala-Cys(S-Farnesyl)OCH3 was synthesized by the condensation of the amine terminal protected decapeptide with the carboxyl terminal farnesylated dipeptide using benzotriazol-l-yloxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP reagent) as the coupling agent. The synthesis of the decapeptide involved 5 + 5 fragment coupling with the BOP reagent and the successful application of 9-fluorenylmethyl ester(OFm) and 9-fluorenylmethoxycarbonyl(Fmoc) groups for the protection of Asp and Lys side chains and Tyr alpha-amine and of phenacyl esters (OPa) for alpha-carboxyl protection. The OFm and Fmoc groups tolerated repeated couplings and were completely stable to zinc powder in acetic acid, a condition under which the OPa group was removed. The synthesis of the nonfarnesylated alpha-factor was accomplished by the coupling of the decapeptide with tetrapeptide (Ala-CysOCH3)2 followed by the deprotection of the OFm and Fmoc groups with piperidine and the cleavage of the disulfide bond with zinc powder in acetic acid. The nonmethylated a-factor was prepared by 10 + 2 fragment coupling using OFm protection of the dipeptide carboxyl group followed by removal of all protecting groups with piperidine. Attempts to saponify a-factor were not successful. The synthetic nonfarnesylated and nonmethylated a-mating pheromones were 100-1000 times less active than the a-factor, indicating that although the methyl ester and the farnesyl group are not essential for biological activity, they are necessary for high potency.     

Total in vitro maturation of the Saccharomyces cerevisiae a-factor lipopeptide mating pheromone

The a-factor mating pheromone, produced by Saccharomyces cerevisiae a haploid cells, is post-translationally modified in a manner analogous to that of the ras proto-oncogene product. A consensus C-terminal amino acid sequence, -CAAX (C is cysteine, A is aliphatic amino acid, and X is any amino acid), is the target of these modifications, which include isoprenylation (essential for Ras function), proteolysis of the -AAX sequence, and carboxy methyl esterification. Recently, the RAM/DPR1 gene product was shown to be a component of the activity responsible for isoprenylation of both Ras and a-factor. In this report, we present an in vitro assay which not only detects a-factor isoprenylation, but also proteolysis and carboxy methyl esterification, and directly demonstrates, biochemically, the order of these processing events. This a-factor maturation assay may prove useful for screening agents which block any of the steps involved in the post-translational modification of the a-factor and Ras -CAAX sequences. Such agents would be potential anti-Ras-related cancer therapeutic drugs.     

Site of action of mating factor in a-mating type cell of Saccharomyces cerevisiae.

The process of the entry of FITC-conjugated mating factor into $\text{a}$-mating type cells of $\text{Saccharomyces}\text{cerevisiae}$ and its concentration into the nucleus were observed. But, when $\text{α}$-mating type cells or diploid cells of $\text{S.}\text{cerevisiae}$ were incubated with the FITC-conjugated mating factor, its adsorption to the cell surface of the test organisms and its incorporation into the cell did not occur. The peptides formed by the cleavage of mating factor by $\text{α}$-mating type cells of $\text{S.}\text{cerevisiae}$ were not adsorbed onto $\text{a}$-mating type cells.     

Lipid synthesis in inositol-starved Saccharomyces cerevisiae

Lipid synthesis was analyzed in an inositol-requiring mutant of Saccharomyces cerevisiae (MC13). Both rates and cellular amounts of [U-14C]acetate incorporation into phospholipids, triacylglycerols, free sterols and steryl esters were elevated in an inositol-starved culture compared to the supplemented control at a time when the deprived culture was losing viability (inositol-less death). The rates at a later time were greatly reduced. During the period when de novo lipid synthesis was high in the starved culture, phospholipid turnover and presumed conversion to triacylglycerols was also accelerated; no differences were apparent in the turnover of the sterol fractions between the two cultures. No change in the fractional percent of ergosterol or of the sterol precursors could be attributed to inositol starvation. The synthesis and maintenance of membrane lipids (phospholipids and free sterols) and their coupling in cellular metabolism are discussed in light of these results.     

Chemo-enzymatic synthesis of the glycosylated alpha-mating factor of Saccharomyces cerevisiae and analysis of its biological activity

The effect of glycosylation on a bioactive peptide was studied using yeast Saccharomyces cerevisiae alpha-mating factor, which is composed of 13 amino acids. In this study, we prepared glycosylated alpha-mating factor by chemo-enzymatic synthesis. At first, N-acetylglucosaminyl alpha-mating factor (Trp-His-Trp-Leu-Gln(GlcNAc)-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr) was chemically synthesized by the solid-phase method. Then, using the transglycosylation activity of Mucor hiemalis endo-beta-N-acetylglucosaminidase, we synthesized glycosylated alpha-mating factor with a glutamine-linked sialo complex type oligosaccharide. The biological activity of alpha-mating factor derivatives was examined by means of a growth arrest assay using secreted-protease-defective a cells of S. cerevisiae. The results showed that the bioactivity of glycosylated alpha-mating factor was lower than that of native alpha-mating factor. However, when sialic acid was removed from the complex type sugar chain of glycosylated alpha-mating factor, its bioactivity was recovered. Glycosylated alpha-mating factor exhibited higher resistance against proteolysis than native alpha-mating factor. It was found that the bioactivity of N-acetylglucosaminyl alpha-mating factor was higher than that of alpha-mating factor. Circular dichroism studies indicated that a slight change in the structure of alpha-mating factor may influence its activity.     

Protein synthesis in germinating Saccharomyces cerevisiae ascospores

The uptake and incorporation of macromolecular precursors in germinating Saccharomyces cerevisiae ascospores were investigated. Addition of cycloheximide at various times during germination revealed that protein synthesis can occur within 20 min after the spores are shifted to glucose-containing media. The time of initiation of uptake and incorporation of several amino acids differed; this can be attributed to differing amino acid pool levels in the spores, as well as differing transport activities. Two-dimensional gel electrophoresis of proteins labeled with [35S]methionine for various 20-min periods after germination began showed at least one protein whose synthesis begins well after the bulk of the proteins.     

beta-1,6-Glucan synthesis in Saccharomyces cerevisiae

beta-1,6-Glucan is an essential fungal-specific component of the Saccharomyces cerevisiae cell wall that interconnects all other wall components into a lattice. Considerable biochemical and genetic effort has been directed at the identification and characterization of the steps involved in its biosynthesis. Structural studies show that the polymer plays a central role in wall structure, attaching mannoproteins via their glycosylphosphatidylinositol (GPI) glycan remnant to beta-1,3-glucan and chitin. Genetic approaches have identified genes that upon disruption result in beta-1,6-glucan defects of varying severity, often with reduced growth or lethality. These gene products have been localized throughout the secretory pathway and at the cell surface, suggesting a possible biosynthetic route. Current structural and genetic data have therefore allowed the development of models to predict biosynthetic events. Based on knowledge of beta-1,3-glucan and chitin synthesis, it is likely that the bulk of beta-1,6-glucan polymer synthesis occurs at the cell surface, but requires key prior intracellular events. However, the activity of most of the identified gene products remain unknown, making it unclear to what extent and how directly they contribute to the synthesis of this polymer. With the recent availability of new tools, reagents and methods (including genomics), the field is poised for a convergence of biochemical and genetic methods to identify and characterize the biochemical steps in the synthesis of this polymer.     

Purification and characterization of protein synthesis initiation factor eIF-4E from the yeast Saccharomyces cerevisiae

A 24 000-dalton protein [yeast eukaryotic initiation factor 4E (eIF-4E)] was purified from yeast Saccharomyces cerevisiae postribosomal supernatant by m7GDP-agarose affinity chromatography. The protein behaves very similarly to mammalian protein synthesis initiation factor eIF-4E with respect to binding to and elution from m7GDP-agarose columns and cross-linking to oxidized reovirus mRNA cap structures. Yeast eIF-4E is required for translation as shown by the strong and specific inhibition of cell-free translation in a yeast extract by a monoclonal antibody directed against yeast eIF-4E.     

Enzymatic synthesis of glutathione using engineered Saccharomyces cerevisiae

Two single gene cassettes, each containing one of the individual gene (γ-glutamylcysteine synthetase gene GSH1 or glutathione synthetase gene GSH2), were constructed under the control of alcohol dehydrogenase (ADH1) promoter and their respective native terminators. The recombinant plasmids constructed with Kan ^r or Hyg ^r as the selective markers and were transformed into Saccharomyces cerevisiae separately and jointly. Three engineered strains, GSH1-enhanced strain S.TS013/GSH1, GSH2-enhanced strain S.TS013/GSH2 and GSH1+GSH2 double-enhanced strain S.TS013/GSH1+GSH2, were constructed. Glutathione production using the recombinant strains to improve was then determined. By the cell dosage proportions of two engineered strains (S.TS013/GSH1, S.TS013/GSH2) and a two-stage reaction, GSH productivity increased by 84 and 59 % over that of the host strain and the S.TS013/GSH1+GSH2 strain, respectively.     

Structure-activity relationships in the dodecapeptide alpha factor of Saccharomyces cerevisiae

Ten analogues of His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr, the dodecapeptide alpha factor of Saccharomyces cerevisiae, were synthesized by conventional solution phase techniques and purified by using high-performance liquid chromatography. The dodecapeptide was also synthesized attached at its carboxyl terminus to poly(ethylene oxide), a macromolecular protecting group. Analogues in which Lys6 or His1 was modified exhibited high biological activity as evidenced by their ability to elicit aberrant morphologies in a cells of S. cerevisiae. These results suggest that neither a free alpha-amine nor a protonatable side chain at position 6 is necessary for biological activity of the dodecapeptide alpha factor. Although Ala2- and Phe2-dodecapeptides were not biologically active, they competed with the natural alpha factor and several active analogues. Thus binding of the alpha factor is not sufficient to elicit a biological response; it appears that the side chain in position 2 is critical for triggering morphological alterations in a cells.     

Fatty acid synthesis in mitochondria from Saccharomyces cerevisiae

The ability of purified mitochondria isolated from S. cerevisiae to synthesize fatty acids and especially very long chain fatty acids (VLCFA) has been investigated. The VLCFA synthesis requires malonyl-CoA as the C2 unit donor and NADPH as the reducing agent. Moreover the yeast mitochondrial elongase is able to accept either exogenous long chain fatty acyl-CoAs as substrates or elongate endogenous substrates. In the latter case, ATP is required for full activity. Besides this important VLCFA formation, the mitochondria from S. cerevisiae were also able to synthesize C16 and C18.     

V-ATPase dysfunction suppresses polyphosphate synthesis in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae accumulates the high levels of inorganic polyphosphates (polyPs) performing in the cells numerous functions, including phosphate and energy storage. The effects of vacuolar membrane ATPase (V-ATPase) dysfunction were studied on polyP accumulation under short-term cultivation in the P_i–excess media after P_i starvation. The addition of bafilomycin A1, a specific inhibitor of V-ATPase, to the medium with glucose resulted in strong inhibition of the synthesis of long-chain polyP and in substantial suppression of short-chain polyP. The addition of bafilomycin to the medium with ethanol resulted in decreased accumulation of high-molecular polyP, while the accumulation of low-molecular polyP was not affected. The levels of polyP synthesis in the mutant strain with a deletion in the vma2 gene encoding a V-ATPase subunit were significantly lower than in the parent strain in the media with glucose and with ethanol. The synthesis of the longest chain polyP was not observed in the mutant cells. The synthesis of only the low-polymer acid-soluble polyP fraction occurred in the cells of the mutant strain. However, the level of polyP1 was nearly tenfold lower than compared to the cells of the parent strain. Both bafilomycin A1 and the mutation in vacuolar ATPase subunit vma2 lead to a considerable decrease of cellular polyP accumulation. Thus, the defects in ΔμH⁺ formation on the vacuolar membrane resulted in the decrease of polyP biosynthesis in S. cerevisiae.     

Alpha-factor-directed synthesis of Bacillus stearothermophilus alpha-amylase in Saccharomyces cerevisiae

Promoter and leader sequence of Bacillus stearothermophilus alpha-amylase gene were removed and the gene was joined in-frame to sequences encoding the leader region of Saccharomyces cerevisiae mating pheromone alpha-factor on plasmid p69A (a hybrid of pBR322 and S. cerevisiae 2-microns plasmid). S. cerevisiae cells were transformed with plasmids containing the hybrid genes, obtaining yeast transformants which exhibit a significant extra-cellular amylolytic activity in solid medium, but not in liquid medium. Levels of alpha-amylase activity in solid medium were found to depend on the mode of fusion of the alpha-amylase gene to the alpha-factor leader region.     

Biotechnological synthesis of water-soluble food-grade polyphosphate with Saccharomyces cerevisiae

Inorganic polyphosphate (polyP) is the polymer of phosphate. Water-soluble polyPs with average chain lengths of 2–40 P-subunits are widely used as food additives and are currently synthesized chemically. An environmentally friendly highly scalable process to biosynthesize water-soluble food-grade polyP in powder form (termed bio-polyP) is presented in this study. After incubation in a phosphate-free medium, generally regarded as safe wild-type baker's yeast (Saccharomyces cerevisiae) took up phosphate and intracellularly polymerized it into 26.5% polyP (as KPO₃, in cell dry weight). The cells were lyzed by freeze-thawing and gentle heat treatment (10 min, 70°C). Protein and nucleic acid were removed from the soluble cell components by precipitation with 50 mM HCl. Two chain length fractions (42 and 11P-subunits average polyP chain length, purity on a par with chemically produced polyP) were obtained by fractional polyP precipitation (Fraction 1 was precipitated with 100 mM NaCl and 0.15 vol ethanol, and Fraction 2 with 1 final vol ethanol), drying, and milling. The physicochemical properties of bio-polyP were analyzed with an enzyme assay, ³¹P nuclear magnetic resonance spectroscopy, and polyacrylamide gel electrophoresis, among others. An envisaged application of the process is phosphate recycling from waste streams into high-value bio-polyP.     

Metabolic control analysis of glycerol synthesis in Saccharomyces cerevisiae

Glycerol, a major by-product of ethanol fermentation by Saccharomyces cerevisiae, is of significant importance to the wine, beer, and ethanol production industries. To gain a clearer understanding of and to quantify the extent to which parameters of the pathway affect glycerol flux in S. cerevisiae, a kinetic model of the glycerol synthesis pathway has been constructed. Kinetic parameters were collected from published values. Maximal enzyme activities and intracellular effector concentrations were determined experimentally. The model was validated by comparing experimental results on the rate of glycerol production to the rate calculated by the model. Values calculated by the model agreed well with those measured in independent experiments. The model also mimics the changes in the rate of glycerol synthesis at different phases of growth. Metabolic control analysis values calculated by the model indicate that the NAD(+)-dependent glycerol 3-phosphate dehydrogenase-catalyzed reaction has a flux control coefficient (C(J)v1) of approximately 0.85 and exercises the majority of the control of flux through the pathway. Response coefficients of parameter metabolites indicate that flux through the pathway is most responsive to dihydroxyacetone phosphate concentration (R(J)DHAP= 0.48 to 0.69), followed by ATP concentration (R(J)ATP = -0.21 to -0.50). Interestingly, the pathway responds weakly to NADH concentration (R(J)NADH = 0.03 to 0.08). The model indicates that the best strategy to increase flux through the pathway is not to increase enzyme activity, substrate concentration, or coenzyme concentration alone but to increase all of these parameters in conjunction with each other.