Nucleotide sequences of Saccharomycopsis fibuligera genes for extracellular beta-glucosidases as expressed in Saccharomyces cerevisiae

We isolated two genes for extracellular beta-glucosidase, BGL1 and BGL2, from the genomic library of the yeast Saccharomycopsis fibuligera. Gene products (BGLI and BGLII) were purified from the culture fluids of Saccharomyces cerevisiae transformed with BGL1 and BGL2, respectively. Molecular weights of BGLI and BGLII were estimated to be 220,000 and 200,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The two beta-glucosidases showed the same enzymatic characteristics, such as thermo-denaturation kinetics and dependencies on pH and temperature, but quite different substrate specificities: BGLI hydrolyzed cellobiose efficiently, but BGLII did not. This result is consistent with the observation that the S. cerevisiae transformant carrying BGL1 fermented cellobiose to ethanol but the transformant carrying BGL2 did not. Southern blot analysis revealed that the two beta-glucosidase genes were derived from Saccharomycopsis fibuligera and that the nucleotide sequences of the two genes are closely related. The complete nucleotide sequences of the two genes were determined. BGL1 and BGL2 encode 876- and 880-amino-acid proteins which were shown to be highly similar to each other. The putative precursors begin with hydrophobic segments that presumably act as signal sequences for secretion. Amino acid analysis of the purified proteins confirmed that BGL1 and BGL2 encode BGLI and BGLII, respectively.     

Common signal transduction system shared by STE2 and STE3 in haploid cells of Saccharomyces cerevisiae: autocrine cell-cycle arrest results from forced expression of STE2

Induction of STE2 expression using the GAL1 promoter both in a wild-type MATalpha strain and in a MATalpha ste3 strain caused transient cell-cycle arrest and changes in morphology ('shmoo'-like phenotype) in a manner similar to alpha cells responding to alpha-factor. In addition, STE2 expressed in a MATalp[ha ste3 mutant allowed the cell to conjugate with alpha cells but at an efficiency lower than that of wil-type alpha cells. This result indicates that signal(s) generated by alpha-factor in alpha cells can be substituted by signal(s) generated by the interaction of alpha-factor with the expressed STE2 product. When STE2 or STE3 was expressed in a matalpha1 strain (insensitive to both alpha- and a-factors), the cell became sensitive to alpha- or a-factor, respectively, and resulted in morphological changes. These results suggest that STE2 and STE3 are the sole determinants for alpha-factor and a-factor sensitivity, respectively, in this strain. On the other hand, expression of STE2 in an a/alpha diploid cell did not affect the alpha-factor insensitive phenotype. Haploid-specific components may be necessary to transduce the alpha-factor signal. These results are consistent with the idea that STE2 encodes an alpha-factor receptor and STE3 encodes an a-factor receptor, and suggest that both alpha- and a-factors may generate an exchangeable signal(s) within haploid cells.     

Nucleotide sequences of Saccharomycopsis fibuligera genes for extracellular beta-glucosidases as expressed in Saccharomyces cerevisiae.

We isolated two genes for extracellular beta-glucosidase, BGL1 and BGL2, from the genomic library of the yeast Saccharomycopsis fibuligera. Gene products (BGLI and BGLII) were purified from the culture fluids of Saccharomyces cerevisiae transformed with BGL1 and BGL2, respectively. Molecular weights of BGLI and BGLII were estimated to be 220,000 and 200,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The two beta-glucosidases showed the same enzymatic characteristics, such as thermo-denaturation kinetics and dependencies on pH and temperature, but quite different substrate specificities: BGLI hydrolyzed cellobiose efficiently, but BGLII did not. This result is consistent with the observation that the S. cerevisiae transformant carrying BGL1 fermented cellobiose to ethanol but the transformant carrying BGL2 did not. Southern blot analysis revealed that the two beta-glucosidase genes were derived from Saccharomycopsis fibuligera and that the nucleotide sequences of the two genes are closely related. The complete nucleotide sequences of the two genes were determined. BGL1 and BGL2 encode 876- and 880-amino-acid proteins which were shown to be highly similar to each other. The putative precursors begin with hydrophobic segments that presumably act as signal sequences for secretion. Amino acid analysis of the purified proteins confirmed that BGL1 and BGL2 encode BGLI and BGLII, respectively.     

Nucleotide sequence of Saccharomyces cerevisiae genes TRP2 and TRP3 encoding bifunctional anthranilate synthase: indole-3-glycerol phosphate synthase

Saccharomyces cerevisiae anthranilate synthase:indole-3-glycerol phosphate synthase is a multifunctional hetero-oligomeric enzyme encoded by genes TRP2 and TRP3. TRP2, encoding anthranilate synthase Component I, was cloned by complementation of a yeast trp2 mutant. The nucleotide sequence of TRP2 as well as that of TRP3 were determined. The deduced anthranilate synthase Component I primary structure from yeast exhibits only limited similarity to that of the corresponding Escherichia coli subunit encoded by trpE. On the other hand, yeast anthranilate synthase Component II and indole-3-glycerol phosphate synthase amino acid sequences from TRP3 are clearly homologous with the corresponding sequences of the E. coli trpG and trpC polypeptide segments and thereby establish the bifunctional structure of TRP3 protein. Based on comparisons of TRP3 amino acid sequence with homologous sequences from E. coli and Neurospora crassa, an 11-amino acid residue connecting segment was identified which fuses the trpG and trpC functions of the bifunctional TRP3 protein chain. These comparisons support the conclusion that the amino acid sequence of connectors in homologous multifunctional enzymes need not be conserved. Connector function is thus not dependent on a specific sequence. Nuclease S1 mapping was used to identify mRNA 5' termini. Heterogeneous 5' termini were found for both TRP2 and TRP3 mRNA. TRP2 and TRP3 5'-flanking regions were analyzed for sequences that might function in regulation of these genes by the S. cerevisiae general amino acid control system. The 9 base pair direct repeat (Hinnebusch, A.G., and Fink, G.R. (1983) J. Biol. Chem. 258, 5238-5247) and inverted repeats were identified in the 5'-flanking sequences of TRP2 and TRP3.     

Isolation of cloned DNA sequences containing ribosomal protein genes from Saccharomyces cerevisiae.

Yeast mRNA enriched for ribosomal protein mRNA was obtained by isolating poly(A)+ small mRNA from small polysomes. A comparison of cell-free translation of this small mRNA and total mRNA, and electrophoresis of the products on two-dimensional gels which resolve most yeast ribosomal proteins, demonstrated that a 5-10 fold enrichment for ribosomal protein mRNA was obtained. One hundred different recombinant DNA molecules possibly containing ribosomal protein genes were selected by differential colony hybridization of this enriched mRNA and unfractionated mRNA to a bank of yeast pMB9 hybrid plasmids. After screening twenty-five of these candidates, five different clones were found which contain yeast ribosomal protein gene sequences. The yeast mRNAs complementary to these five plasmids code for 35S-methionine-labeled polypeptides which co-migrate on two-dimensional gels with yeast ribosomal proteins. Consistent with previous studies on ribosomal protein mRNAs, the amounts of mRNA complementary to three of these cloned genes are controlled by the RNA2 locus. Although two of the five clones contain more than one yeast gene, none contain more than one identifiable ribosomal protein gene. Thus there is no evidence for "tight" linkage of yeast ribosomal protein genes. Two of the cloned ribosomal protein genes are single-copy genes, whereas two other cloned sequences contain two different copies of the same ribosomal protein gene. The fifth plasmid contains sequences which are repeated in the yeast genome, but it is not known whether any or all of the ribosomal protein gene on this clone contains repetitive DNA.     

Glucose regulation of Saccharomyces cerevisiae cell cycle genes

Nutrient-limited Saccharomyces cerevisiae cells rapidly resume proliferative growth when transferred into glucose medium. This is preceded by a rapid increase in CLN3, BCK2, and CDC28 mRNAs encoding cell cycle regulatory proteins that promote progress through Start. We have tested the ability of mutations in known glucose signaling pathways to block glucose induction of CLN3, BCK2, and CDC28. We find that loss of the Snf3 and Rgt2 glucose sensors does not block glucose induction, nor does deletion of HXK2, encoding the hexokinase isoenzyme involved in glucose repression signaling. Rapamycin blockade of the Tor nutrient sensing pathway does not block the glucose response. Addition of 2-deoxy glucose to the medium will not substitute for glucose. These results indicate that glucose metabolism generates the signal required for induction of CLN3, BCK2, and CDC28. In support of this conclusion, we find that addition of iodoacetate, an inhibitor of the glyceraldehyde-3-phosphate dehydrogenase step in yeast glycolysis, strongly downregulates the levels CLN3, BCK2, and CDC28 mRNAs. Furthermore, mutations in PFK1 and PFK2, which encode phosphofructokinase isoforms, inhibit glucose induction of CLN3, BCK2, and CDC28. These results indicate a link between the rate of glycolysis and the expression of genes that are critical for passage through G₁.     

The a-factor transporter (STE6 gene product) and cell polarity in the yeast Saccharomyces cerevisiae

STE6 gene product is required for secretion of the lipopeptide mating pheromone a-factor by Saccharomyces cerevisiae MATa cells. Radiolabeling and immunoprecipitation, either with specific polyclonal antibodies raised against a TrpE-Ste6 fusion protein or with mAbs that recognize c-myc epitopes in fully functional epitope-tagged Ste6 derivatives, demonstrated that Ste6 is a 145-kD phosphoprotein. Subcellular fractionation, various extraction procedures, and immunoblotting showed that Ste6 is an intrinsic plasma membrane- associated protein. The apparent molecular weight of Ste6 was unaffected by tunicamycin treatment, and the radiolabeled protein did not bind to concanavalin A, indicating that Ste6 is not glycosylated and that glycosylation is not required either for its membrane delivery or its function. The amino acid sequence of Ste6 predicts two ATP- binding folds; correspondingly, Ste6 was photoaffinity-labeled specifically with 8-azido-[alpha-32P]ATP. Indirect immunofluorescence revealed that in exponentially growing MATa cells, the majority of Ste6 showed a patchy distribution within the plasma membrane, but a significant fraction was found concentrated in a number of vesicle-like bodies subtending the plasma membrane. In contrast, in MATa cells exposed to the mating pheromone alpha-factor, which markedly induced Ste6 production, the majority of Ste6 was incorporated into the plasma membrane within the growing tip of the elongating cells. The highly localized insertion of this transporter may establish pronounced anisotropy in a-factor secretion from the MATa cell, and thereby may contribute to the establishment of the cell polarity which restricts partner selection and cell fusion during mating to one MAT alpha cell.