Differential utilisation of sulfur compounds for H(2)S liberation by nitrogen-starved wine yeasts

Hydrogen sulfide (H2S) liberation by Saccharomyces cerevisiae wine yeast was studied as a function of different inorganic sulfurous precursors using a model winemaking system. In a medium analogous to grape juice, and following nitrogen starvation of the yeast culture, a combination of 5 mM sulfate and 300 μM sulfite was more potent than 5 mM sulfate alone as a substrate for H2S liberation. Sulfate plus sulfite allowed H2S liberation in greater amounts, at higher rates and over a longer period than sulfate alone, by most of the five yeast strains examined. Nevertheless, a sulfate-only medium still supported the liberation of between 11 and 86% of the H2S observed in the sulfate plus sulfite medium. After nitrogen depletion of the medium, sulfate uptake by yeast cells was stable for at least 7 h and even increased in a cycloheximide-sensitive manner by ca 35%. The relatively limited formation of H2S from sulfate is therefore not attributable to losses of sulfate transport by the culture as might occur through elevated permease turnover. Similarly, an inhibition of sulfate transport could not account for the lesser formation of H2S from sulfate because cultures starved of nitrogen in the presence of sulfate continued to accumulate sulfate in excess of the rates of H2S liberation. Instead, sulfate deprivation/supplementation experiments implied that limited utilisation of sulfate was due to an inhibition of sulfate reduction to sulfide. Experiments also highlighted a contribution by intracellular sulfur pools of between 35 and 70% to the total H2S liberated from sulfate by nitrogen starved yeasts. As a component of this pool, glutathione was a precursor for 40% of the H2S liberated from sulfate-containing medium.Hallinan, Christopher P. ; Saul, David J. ; Jiranek, Vladimi

Yeast Glycogen Synthase Kinase-3 Activates Msn2p-dependent Transcription of Stress Responsive Genes

The yeast Saccharomyces cerevisiae has four genes, MCK1, MDS1 (RIM11), MRK1, and YOL128c, that encode homologues of mammalian glycogen synthase kinase 3 (GSK-3). A gsk-3 null mutant in which these four genes are disrupted showed growth defects on galactose medium. We isolated several multicopy suppressors of this growth defect. Two of them encoded Msn2p and phosphoglucomutase (PGM). Msn2p is a transcription factor that binds to the stress-response element (STRE). PGM is an enzyme that interconverts glucose-1 phosphate and glucose-6 phosphate and is regulated by Msn2p at the transcriptional level. Expression of the mRNAs of PGM2 and DDR2, whose promoter regions possess STRE sequences, on induction by heat shock or salt stress was reduced not only in an msn2 msn4 (msn2 homologue) double mutant but also in the gsk-3 null mutant. STRE-dependent transcription was greatly inhibited in the gsk-3 null mutant or mck1 mds1 double mutant, and this phenotype was suppressed by the expression of Mck1p but not of a kinase-inactive form of Mck1p. Although Msn2p accumulated in the nucleus of the gsk-3 null mutant as well as in the wild-type strain under various stress conditions, its STRE-binding activity was reduced in extracts prepared from the gsk-3 null mutant or mck1 mds1 double mutant. These results suggest that yeast GSK-3 promotes formation of a complex between Msn2p and DNA, which is required for the proper response to different forms of stress. Because neither Msn2p–GSK-3 complex formation nor GSK-3–dependent phosphorylation of Msn2p could be detected, the regulation of Msn2p by GSK-3 may be indirect

A mechanistic classification of clinical phenotypes in neuroblastoma

Neuroblastoma is a pediatric tumor of the sympathetic nervous system. Its clinical course ranges from spontaneous tumor regression to fatal progression. To investigate the molecular features of the divergent tumor subtypes, we performed genome sequencing on 416 pretreatment neuroblastomas and assessed telomere maintenance mechanisms in 208 of these tumors. We found that patients whose tumors lacked telomere maintenance mechanisms had an excellent prognosis, whereas the prognosis of patients whose tumors harbored telomere maintenance mechanisms was substantially worse. Survival rates were lowest for neuroblastoma patients whose tumors harbored telomere maintenance mechanisms in combination with RAS and/or p53 pathway mutations. Spontaneous tumor regression occurred both in the presence and absence of these mutations in patients with telomere maintenance-negative tumors. On the basis of these data, we propose a mechanistic classification of neuroblastoma that may benefit the clinical management of patients

A Yeast t-SNARE Involved in Endocytosis

The ORF YOL018c (TLG2) of Saccharomyces cerevisiae encodes a protein that belongs to the syntaxin protein family. The proteins of this family, t-SNAREs, are present on target organelles and are thought to participate in the specific interaction between vesicles and acceptor membranes in intracellular membrane trafficking. TLG2 is not an essential gene, and its deletion does not cause defects in the secretory pathway. However, its deletion in cells lacking the vacuolar ATPase subunit Vma2p leads to loss of viability, suggesting that Tlg2p is involved in endocytosis. In tlg2Δ cells, internalization was normal for two endocytic markers, the pheromone α-factor and the plasma membrane uracil permease. In contrast, degradation of α-factor and uracil permease was delayed in tlg2Δ cells. Internalization of positively charged Nanogold shows that the endocytic pathway is perturbed in the mutant, which accumulates Nanogold in primary endocytic vesicles and shows a greatly reduced complement of early endosomes. These results strongly suggest that Tlg2p is a t-SNARE involved in early endosome biogenesis