Piecemeal microautophagy of nucleus in Saccharomyces cerevisiae

Nucleus-vacuole (NV) junctions in Saccharomyces cerevisiae are formed through specific interactions between Vac8p on the vacuole membrane and Nvj1p in the nuclear envelope. Herein, we report that NV junctions in yeast promote piecemeal microautophagy of the nucleus (PMN). During PMN, teardrop-like blebs are pinched from the nucleus, released into the vacuole lumen, and degraded by soluble hydrolases. PMN occurs in rapidly dividing cells but is induced to higher levels by carbon and nitrogen starvation and is under the control of the Tor kinase nutrient-sensing pathway. Confocal and biochemical assays demonstrate that Nvj1p is degraded in a PMN-dependent manner. PMN occurs normally in apg7-delta cells and is, therefore, not dependent on macroautophagy. Transmission electron microscopy reveals that portions of the granular nucleolus are often sequestered into PMN structures. These results introduce a novel mode of selective microautophagy that targets nonessential components of the yeast nucleus for degradation and recycling in the vacuole.     

Measuring piecemeal microautophagy of the nucleus in Saccharomyces cerevisiae

Piecemeal microautophagy of the nucleus (PMN) selectively removes and degrades small fragments of the Saccharomyces cerevisiae nucleus. Inter-organelle contact sites called nucleus-vacuole (NV) junctions determine the selectivity of PMN by establishing a platform for the biogenesis of PMN blebs and vesicles. PMN structures can be observed by fluorescence microscopy using GFP-tagged reporters; however, this approach is best supported with quantitative immunoblot assays of PMN-specific cargo degradation. Together, these assays should facilitate the further study of this fascinating but poorly understood autophagic process in different genetic backgrounds, physiological states, and environmental conditions.     

苹果酸降解相关基因在酿酒酵母中的表达

微生物降酸是现代葡萄酒酿造重要工艺。将裂殖酵母苹果酸通透酶基因(mae1)和苹果酸酶基因(mae2)克隆到酿酒酵母中,构建了苹果酸酒精酵母;将mae1基因和乳酸乳球菌的苹果酸乳酸酶基因(mleS)克隆到酿酒酵母中,构建了苹果酸乳酸酵母。构建的酵母重组子能够有效地分解发酵基质中的苹果酸。     

Degradation of polygalacturonic acid by Saccharomyces cerevisiae

Thirty-three strains of Saccharomyces cereuisiae were tested for the ability to degrade polygalacturonic acid. Nine strains degraded polygalacturonic acid during growth in liquid or on solid medium supplemented with glucose. This ability of certain strains of S. cereuisiae may be important in the fermentation plant products.     

Clustering of the Genes for Allantoin Degradation in Saccharomyces cerevisiae

We have shown that allantoin degradation in Saccharomyces cerevisiae proceeds exclusively through the intermediate formation of allantoic acid, urea, and allophanic acid. The number of reactions between allantoic acid and urea, however, remains obscure owing to our inability to isolate a mutant defective in ureidoglycolate hydrolase. Structural genes for the enzymes, allantoinase (dal1) and allantoicase (dal2) are located on chromosome IX promixal to the centromere in the order dal1-dal2-lysl.     

Genome-wide analysis of mRNA lengths in Saccharomyces cerevisiae

Background  

Although the protein-coding sequences in the Saccharomyces cerevisiae genome have been studied and annotated extensively, much less is known about the extent and characteristics of the untranslated regions of yeast mRNAs.     

The half-life of mRNA in Saccharomyces cerevisiae.

Summary The decay kinetics of mRNA was studied in a yeast temperature-sensitive mutant, ts136, which is defective in cytoplasmic RNA production at 37° C. The disappearance of the synthetic capacity of mRNA was determined by withdrawing equal volumes of ts136 cell culture and pulse-labelling with [³⁵S]methionine at various time intervals after the shift to 37° C from 23° C. The synthesized proteins were separated on a two-dimensional gel electrophoretic system and then quantitatively analyzed for their incorporated radioactivities by scintillation counting. Our results show that yeast mRNAs have divergent functional half-lives ranging from 4.5 to 41 min, with an average value of 22 min. Each mRNA exhibits a simple exponential decay with its own characteristic decay pattern. Of the approximately 500 major polypeptides made by yeast cells, which are detectable on autoradiograms of the gels, 80 were arbitrarily selected and the mRNAs coding for those polypeptides were examined for their decay kinetics.     

Degradation of the hexose transporter Hxt5p in Saccharomyces cerevisiae

BACKGROUND INFORMATION: Hxt5p is a member of a multigene family of hexose transporter proteins which translocate glucose across the plasma membrane of the yeast Saccharomyces cerevisiae. In contrast with other major hexose transporters of this family, Hxt5p expression is regulated by the growth rate of the cells and not by the external glucose concentration. Furthermore, Hxt5p is the only glucose transporter expressed during stationary phase. These observations suggest a different role for Hxt5p in S. cerevisiae. Therefore we studied the metabolism and localization of Hxt5p in more detail. RESULTS AND CONCLUSIONS: Inhibition of HXT5 expression in stationary-phase cells by the addition of glucose, which increases the growth rate, led to a decrease in the amount of Hxt5 protein within a few hours. Addition of glucose to stationary-phase cells resulted in a transient phosphorylation of Hxt5p on serine residues, but no ubiquitination was detected. The decrease in Hxt5p levels is caused by internalization of the protein, as observed by immunofluorescence microscopy. In stationary-phase cells, Hxt5p was localized predominantly at the cell periphery and upon addition of glucose to the cells the protein translocated to the cell interior. Electron microscopy demonstrated that the internalized Hxt5p-HA (haemagglutinin) protein was localized to small vesicles, multivesicular bodies and the vacuole. These results suggest that internalization and degradation of Hxt5p in the vacuole occur in an ubiquitination-independent manner via the endocytic pathway.     

mRNA structures influencing translation in the yeast Saccharomyces cerevisiae.

The mRNA sequence and structures that modify and are required for translation of iso-1-cytochrome c in the yeast Saccharomyces cerevisiae were investigated with sets of CYC1 alleles having alterations in the 5' leader region. Measurements of levels of CYC1 mRNA and iso-1-cytochrome c in strains having single copies of altered alleles with nested deletions led to the conclusion that there is no specific sequence adjacent to the AUG initiator codon required for efficient translation. However, the nucleotides preceding the AUG initiator codon at positions -1 and -3 slightly modified the efficiency of translation to an order of preference similar to that found in higher cells. In contrast to large effects observed in higher eucaryotes, the magnitude of this AUG context effect in S. cerevisiae was only two- to threefold. Furthermore, introduction of hairpin structures in the vicinity of the AUG initiator codon inhibited translation, with the degree of inhibition related to the stability and proximity of the hairpin. These results with S. cerevisiae and published findings on other organisms suggest that translation in S. cerevisiae is more sensitive to secondary structures than is translation in higher eucaryotes.     

Degradation of Specific Nuclear Proteins Occurs in the Cytoplasm in Saccharomyces cerevisiae

The ubiquitin/proteasome system has been characterized extensively, although the site of nuclear substrate turnover has not been established definitively. We report here that two well-characterized nuclear proteins are stabilized in nuclear export mutants in Saccharomyces cerevisiae. The requirement for nuclear export defines a new regulatory step in intracellular proteolysis.