Characterization of the divergent sacBK and sacAR operons, involved in sucrose utilization by Lactococcus lactis.
The divergently transcribed sacBK and sacAR operons, which are involved in the utilization of sucrose by Lactococcus lactis NZ9800, were examined by transcriptional and gene inactivation studies. Northern analyses of RNA isolated from cells grown at the expense of different carbon sources revealed three sucrose-inducible transcripts: one of 3.2 kb containing sacB and sacK, a second of 3.4 kb containing sacA and sacR, and a third of 1.8 kb containing only sacR. The inactivation of the sacR gene by replacement recombination resulted in the constitutive transcription of the sacBK and sacAR operons in the presence of different carbon sources, indicating that SacR acts as a repressor of transcription. (+info)
Ligand recognition and domain structure of Vps10p, a vacuolar protein sorting receptor in Saccharomyces cerevisiae.
Vp10p is a receptor that sorts several different vacuolar proteins by cycling between a late Golgi compartment and the endosome. The cytoplasmic tail of Vps10p is necessary for the recycling, whereas the lumenal domain is predicted to interact with the soluble ligands. We have studied ligand binding to Vps10p by introducing deletions in the lumenal region. This region contains two domains with homology to each other. Domain 2 binds carboxypeptidase Y (CPY), proteinase A (PrA) and hybrids of these proteases with invertase. Moreover, we show that aminopeptidase Y (APY) is a ligand of Vps10p. The native proteases compete for binding to domain 2. Binding of CPY(156)-invertase or PrA(137)-invertase, on the other hand, do not interfere with binding of CPY to Vps10p. Furthermore, the Q24RPL27 sequence known to be important for vacuolar sorting of CPY, is of little importance in the Vps10p-dependent sorting of CPY-invertase. Apparently, domain 2 contains two different binding sites; one for APY, CPY and PrA, and one for CPY-invertase and PrA-invertase. The latter interaction seems not to be sequence specific, and we suggest that an unfolded structure in these ligands is recognized by Vps10p. (+info)
Novel alleles of yeast hexokinase PII with distinct effects on catalytic activity and catabolite repression of SUC2.
In the yeast Saccharomyces cerevisiae, glucose or fructose represses the expression of a large number of genes. The phosphorylation of glucose or fructose is catalysed by hexokinase PI (Hxk1), hexokinase PII (Hxk2) and a specific glucokinase (Glk1). The authors have shown previously that either Hxk1 or Hxk2 is sufficient for a rapid, sugar-induced disappearance of catabolite-repressible mRNAs (short-term catabolite repression). Hxk2 is specifically required and sufficient for long-term glucose repression and either Hxk1 or Hxk2 is sufficient for long-term repression by fructose. Mutants lacking the TPS1 gene, which encodes trehalose 6-phosphate synthase, can not grow on glucose or fructose. In this study, suppressor mutations of the growth defect of a tps1delta hxk1delta double mutant on fructose were isolated and identified as novel HXK2 alleles. All six alleles studied have single amino acid substitutions. The mutations affected glucose and fructose phosphorylation to a different extent, indicating that Hxk2 binds glucose and fructose via distinct mechanisms. The mutations conferred different effects on long- and short-term repression. Two of the mutants showed very similar defects in catabolite repression, despite large differences in residual sugar-phosphorylation activity. The data show that the long- and short-term phases of catabolite repression can be dissected using different hexokinase mutations. The lack of correlation between in vitro catalytic hexokinase activity, in vivo sugar phosphate accumulation and the establishment of catabolite repression suggests that the production of sugar phosphate is not the sole role of hexokinase in repression. Using the set of six hxk2 mutants it was shown that there is a good correlation between the glucose-induced cAMP signal and in vivo hexokinase activity. There was no correlation between the cAMP signal and the short- or long-term repression of SUC2, arguing against an involvement of cAMP in either stage of catabolite repression. (+info)
Evidence for the involvement of the Glc7-Reg1 phosphatase and the Snf1-Snf4 kinase in the regulation of INO1 transcription in Saccharomyces cerevisiae.
Binding of the TATA-binding protein (TBP) to the promoter is a pivotal step in RNA polymerase II transcription. To identify factors that regulate TBP, we selected for suppressors of a TBP mutant that exhibits promoter-specific defects in activated transcription in vivo and severely reduced affinity for TATA boxes in vitro. Dominant mutations in SNF4 and recessive mutations in REG1, OPI1, and RTF2 were isolated that specifically suppress the inositol auxotrophy of the TBP mutant strains. OPI1 encodes a repressor of INO1 transcription. REG1 and SNF4 encode regulators of the Glc7 phosphatase and Snf1 kinase, respectively, and have well-studied roles in glucose repression. In two-hybrid assays, one SNF4 mutation enhances the interaction between Snf4 and Snf1. Suppression of the TBP mutant by our reg1 and SNF4 mutations appears unrelated to glucose repression, since these mutations do not alleviate repression of SUC2, and glucose levels have little effect on INO1 transcription. Moreover, mutations in TUP1, SSN6, and GLC7, but not HXK2 and MIG1, can cause suppression. Our data suggest that association of TBP with the TATA box may be regulated, directly or indirectly, by a substrate of Snf1. Analysis of INO1 transcription in various mutant strains suggests that this substrate is distinct from Opi1. (+info)
Yeast VSM1 encodes a v-SNARE binding protein that may act as a negative regulator of constitutive exocytosis.
We have screened for proteins that interact with v-SNAREs of the late secretory pathway in the yeast Saccharomyces cerevisiae. A novel protein, designated Vsm1, binds tightly to the Snc2 v-SNARE in the two-hybrid system and can be coimmunoprecipitated with Snc1 or Snc2 from solubilized yeast cell extracts. Disruption of the VSM1 gene results in an increase of proteins secreted into the medium but does not affect the processing or secretion of invertase. In contrast, VSM1 overexpression in cells which bear a temperature-sensitive mutation in the Sec9 t-SNARE (sec9-4 cells) results in the accumulation of non-invertase-containing low-density secretory vesicles, inhibits cell growth and the secretion of proteins into the medium, and blocks rescue of the temperature-sensitive phenotype by SNC1 overexpression. Yet, VSM1 overexpression does not affect yeast bearing a sec9-7 allele which, in contrast to sec9-4, encodes a t-SNARE protein capable of forming a stable SNARE complex in vitro at restrictive temperatures. On the basis of these results, we propose that Vsm1 is a novel v-SNARE-interacting protein that appears to act as negative regulator of constitutive exocytosis. Moreover, this regulation appears specific to one of two parallel exocytic paths which are operant in yeast cells. (+info)
Glycosylation of the overlapping sequons in yeast external invertase: effect of amino acid variation on site selectivity in vivo and in vitro.
Yeast invertase contains 14 sequons, all of which are glycosylated to varying degrees except for sequon 5 which is marginally glycosylated, if at all. This sequon overlaps with sequon 4 in a sequence consisting of Asn92-Asn93-Thr94-Ser95(Reddy et al., 1988, J. Biol. Chem., 263, 6978-6985). To determine whether glycosylation at Asn93is sterically hindered by the oligosaccharide on Asn92, the latter amino acid was converted to a glutamine residue by site-directed mutagenesis of the SUC2 gene in a plasmid vector which was expressed in Saccharomyces cerevisiae. A glycopeptide encompassing sequons 3 through 6 was purified from a tryptic digest of the mutagenized invertase and sequenced by Edman degradation, which revealed that Asn93 of sequon 5 contained very little, if any, carbohydrate, despite the elimination of sequon 4. When Ser and Thr were inverted to yield Asn-Asn-Ser-Thr carbohydrate was associated primarily with the second sequon, in agreement with numerous studies indicating that Asn-X-Thr is preferred to Asn-X-Ser as an oligosaccharide acceptor. However, when the invertase overlapping sequons were converted to Asn-Asn-Ser-Ser, both sequons were clearly glycosylated, with the latter sequon predominating. These findings rule out steric hindrance as a factor involved in preventing the glycosylation of sequon 5 in invertase. Comparable results were obtained using an in vitro system with sequon-containing tri- and tetrapeptides acceptors, in addition to larger oligosaccharide acceptors. (+info)
Std1 and Mth1 proteins interact with the glucose sensors to control glucose-regulated gene expression in Saccharomyces cerevisiae.
The Std1 protein modulates the expression of glucose-regulated genes, but its exact molecular role in this process is unclear. A two-hybrid screen for Std1-interacting proteins identified the hydrophilic C-terminal domains of the glucose sensors, Snf3 and Rgt2. The homologue of Std1, Mth1, behaves differently from Std1 in this assay by interacting with Snf3 but not Rgt2. Genetic interactions between STD1, MTH1, SNF3, and RGT2 suggest that the glucose signaling is mediated, at least in part, through interactions of the products of these four genes. Mutations in MTH1 can suppress the raffinose growth defect of a snf3 mutant as well as the glucose fermentation defect present in cells lacking both glucose sensors (snf3 rgt2). Genetic suppression by mutations in MTH1 is likely to be due to the increased and unregulated expression of hexose transporter genes. In media lacking glucose or with low levels of glucose, the hexose transporter genes are subject to repression by a mechanism that requires the Std1 and Mth1 proteins. An additional mechanism for glucose sensing must exist since a strain lacking all four genes (snf3 rgt2 std1 mth1) is still able to regulate SUC2 gene expression in response to changes in glucose concentration. Finally, studies with green fluorescent protein fusions indicate that Std1 is localized to the cell periphery and the cell nucleus, supporting the idea that it may transduce signals from the plasma membrane to the nucleus. (+info)
A role for Tlg1p in the transport of proteins within the Golgi apparatus of Saccharomyces cerevisiae.
Members of the syntaxin protein family participate in the docking-fusion step of several intracellular vesicular transport events. Tlg1p has been identified as a nonessential protein required for efficient endocytosis as well as the maintenance of normal levels of trans-Golgi network proteins. In this study we independently describe Tlg1p as an essential protein required for cell viability. Depletion of Tlg1p in vivo causes a defect in the transport of the vacuolar protein carboxypeptidase Y through the early Golgi. Temperature-sensitive (ts) mutants of Tlg1p also accumulate the endoplasmic reticulum/cis-Golgi form of carboxypeptidase Y at the nonpermissive temperature (38 degrees C) and exhibit underglycosylation of secreted invertase. Overexpression of Tlg1p complements the growth defect of vti1-11 at the nonpermissive temperature, whereas incomplete complementation was observed with vti1-1, further suggesting a role for Tlg1p in the Golgi apparatus. Overexpression of Sed5p decreases the viability of tlg1 ts mutants compared with wild-type cells, suggesting that tlg1 ts mutants are more susceptible to elevated levels of Sed5p. Tlg1p is able to bind His6-tagged Sec17p (yeast alpha-SNAP) in a dose-dependent manner and enters into a SNARE complex with Vti1p, Tlg2p, and Vps45p. Morphological analyses by electron microscopy reveal that cells depleted of Tlg1p or tlg1 ts mutants incubated at the restrictive temperature accumulate 40- to 50-nm vesicles and experience fragmentation of the vacuole. (+info)