Recessive nonsense-suppression in yeast Saccharomyces cerevisiae

Summary The shift of recessive suppressor mutant of yeast Saccharomyces cerevisiae from permissive to restrictive conditions is accompanied by polysome decay and accumulation of 80 S ribosomes (Smirnov et al., 1976). In this paper some properties of 80 S ribosomes are studied. It is demonstrated that polysome decay under non-permissive conditions is not the consequence of the impairement of RNA synthesis. More than 70% of 80 S ribosomes accumulated under non-permissive conditions contain bound peptidyl-tRNAs localized in P-ribosomal site. tRNA moiety of bound peptidyl-tRNA is able to accept all 20 natural amino acids after chemical deacylation. Therefore it is not a specific isoacceptor species but rather total tRNA that is bound to ribosomes. The polypeptide residues of these peptidyl-tRNAs are heterogeneous in size. Their molecular weights are comparable with the molecular weights of the completed polypeptides. Some of the 80 S ribosomes accumulated under non-permissive conditions contain poly-A RNA. In conclusion, possible mechanism of the impairement of translation under non-permissive conditions in recessive suppressor strain is discussed.     

Recessive nonsense-suppression in yeast: Involvement of 60S ribosomal subunit

Summary The ribosomal protein patterns of recessive suppressor strain and parent strain of Saccharomyces cerevisiae were analyzed by two-dimensional polyacrylamide gel electrophoresis. About 30 protein spots were found for ribosomal proteins of small subunit for both mutant and parent strain. These patterns do not differ from each other neither in intensity of staining, nor in mobility of spots. 41 protein spots were found in electrophoregrams of 60S ribosomal proteins both from parent strain and recessive suppressor strain. The electrophoretic picture of the 60S proteins from the parent and mutant strains is similar except the intensity of staining of the L30 spot. This protein is present in 60S subunit of suppressor strain and completely absent or only weakly stained on electrophoregrams of ribosomal proteins of parent strain. The possible relationships between the content of L30 protein and the mechanism of recessive suppression in yeast are discussed.     

Iron-reductases in the yeast Saccharomyces cerevisiae

Several NAD(P)H-dependent ferri-reductase activities were detected in sub-cellular extracts of the yeast Saccharomyces cerevisiae. Some were induced in cells grown under iron-deficient conditions. At least two cytosolic iron-reducing enzymes having different substrate specificities could contribute to iron assimilation in vivo. One enzyme was purified to homogeneity: it is a flavoprotein (FAD) of 40 kDa that uses NADPH as electron donor and Fe(III)-EDTA as artificial electron acceptor. Isolated mitochondria reduced a variety of ferric chelates, probably via an 'external' NADH dehydrogenase, but not the siderophore ferrioxamine B. A plasma membrane-bound ferri-reductase system functioning with NADPH as electron donor and FMN as prosthetic group was purified 100-fold from isolated plasma membranes. This system may be involved in the reductive uptake of iron in vivo.     

Aquaporins in Saccharomyces cerevisiae wine yeast

AQY1 and AQY2 were sequenced from five commercial and five native wine yeasts. Of these, two AQY1 alleles from UCD 522 and UCD 932 were identified that encoded three or four amino-acid changes, respectively, compared with the Sigma1278b sequence. Oocytes expressing these AQY1 alleles individually exhibited increased water permeability vs. water-injected oocytes, whereas oocytes expressing the AQY2 allele from UCD 932 did not show an increase, as expected, owing to an 11 bp deletion. Wine strains lacking Aqy1p did not show a decrease in spore fitness or enological aptitude under stressful conditions, limited nitrogen, or increased temperature. The exact role of aquaporins in wine yeasts remains unclear.     

Sporulation in the budding yeast Saccharomyces cerevisiae

In response to nitrogen starvation in the presence of a poor carbon source, diploid cells of the yeast Saccharomyces cerevisiae undergo meiosis and package the haploid nuclei produced in meiosis into spores. The formation of spores requires an unusual cell division event in which daughter cells are formed within the cytoplasm of the mother cell. This process involves the de novo generation of two different cellular structures: novel membrane compartments within the cell cytoplasm that give rise to the spore plasma membrane and an extensive spore wall that protects the spore from environmental insults. This article summarizes what is known about the molecular mechanisms controlling spore assembly with particular attention to how constitutive cellular functions are modified to create novel behaviors during this developmental process. Key regulatory points on the sporulation pathway are also discussed as well as the possible role of sporulation in the natural ecology of S. cerevisiae.     

Damage-induced recombination in the yeast Saccharomyces cerevisiae

Prokaryotic and eukaryotic cells have developed a network of DNA repair systems that restore genomic integrity following DNA damage from endogenous and exogenous genotoxic sources. One of the mechanisms used to repair damaged chromosomes is genetic recombination, in which information present as a second chromosomal copy is used to repair a damaged region of the genome. In this review, I summarized what is known about the molecular and cellular mechanisms by which various DNA-damaging agents induce recombination in yeast. The yeast Saccharomyces cerevisiae has served as an excellent model organism to study the induction of recombination. It has helped to define the basic phenomenology and to isolate the genes involved in the process. Given the evolutionary conservation of the various DNA repair systems in eukaryotes, it is likely that the knowledge gathered about induced recombination in yeast is applicable to mammalian cells and thus to humans. Many carcinogens are known to induce recombination and to cause chromosomal rearrangements. An understanding of the mechanisms, by which genotoxic agents cause increased levels of recombination will have important consequences for the treatment of cancer, and for the assessment of risks arising from exposure to genotoxic agents in humans.     

Exopolyphosphatases of the yeast Saccharomyces cerevisiae

Separate compartments of the yeast cell possess their own exopolyphosphatases differing from each other in their properties and dependence on culture conditions. The low-molecular-mass exopolyphosphatases of the cytosol, cell envelope, and mitochondrial matrix are encoded by the PPX1 gene, while the high-molecular-mass exopolyphosphatase of the cytosol and those of the vacuoles, mitochondrial membranes, and nuclei are presumably encoded by their own genes. Based on recent works, a preliminary classification of the yeast exopolyphosphatases is proposed.     

Ubiquitin regulates TORC1 in yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae the TOR complex 1 (TORC1) controls many growth‐related cellular processes and is essential for cell growth and proliferation. Macrolide antibiotic rapamycin, in complex with a cytosol protein named FKBP12, specifically inhibits TORC1, causing growth arrest. The FKBP12‐rapamycin complex interferes with TORC1 function by binding to the FRB domain of the TOR proteins. In an attempt to understand the role of the FRB domain in TOR function, we identified a single point mutation (Tor2^W2041R) in the FRB domain of Tor2 that renders yeast cells rapamycin resistant and temperature sensitive. At the permissive temperature, the Tor2 mutant protein is partially defective for binding with Kog1 and TORC1 is impaired for membrane association. At the restrictive temperature, Kog1 but not the Tor2 mutant protein, is rapidly degraded. Overexpression of ubiquitin stabilizes Kog1 and suppresses the growth defect associated with the tor2 mutant at the nonpremissive temperature. We find that ubiquitin binds non‐covalently to Kog1, prevents Kog1 from degradation and stabilizes TORC1. Our data reveal a unique role for ubiquitin in regulation of TORC1 and suggest that Kog1 requires association with the Tor proteins for stabilization.     

Stationary phase in the yeast Saccharomyces cerevisiae.

Growth and proliferation of microorganisms such as the yeast Saccharomyces cerevisiae are controlled in part by the availability of nutrients. When proliferating yeast cells exhaust available nutrients, they enter a stationary phase characterized by cell cycle arrest and specific physiological, biochemical, and morphological changes. These changes include thickening of the cell wall, accumulation of reserve carbohydrates, and acquisition of thermotolerance. Recent characterization of mutant cells that are conditionally defective only for the resumption of proliferation from stationary phase provides evidence that stationary phase is a unique developmental state. Strains with mutations affecting entry into and survival during stationary phase have also been isolated, and the mutations have been shown to affect at least seven different cellular processes: (i) signal transduction, (ii) protein synthesis, (iii) protein N-terminal acetylation, (iv) protein turnover, (v) protein secretion, (vi) membrane biosynthesis, and (vii) cell polarity. The exact nature of the relationship between these processes and survival during stationary phase remains to be elucidated. We propose that cell cycle arrest coordinated with the ability to remain viable in the absence of additional nutrients provides a good operational definition of starvation-induced stationary phase.     

cAMP-dependent phosphoprotein changes in the yeast Saccharomyces cerevisiae

Abstract cAMP-dependent phosphoprotein changes were determined using 1-dimensional SDS-gel electrophoresis in a cAMP-requiring yeast mutant ( Saccharomyces cerevisiae AM18). During cAMP starvation, the yeast cells accumulated 3 ³²P-labeled bands with M _r/ 72000, 54000, and 37000. The M _r/ 72000 protein was the most prominent phosphorylated protein. After the readdition of cAMP, these phosphoproteins lost their ³²P-label while phosphoproteins with M _r/ 76000, 65000, 56000 and 30000 were accumulated. Similar phosphoprotein changes were also detected in cdc35 at the nonpermissive temperature, but not in wildtype (A363A) or cdc7 strains of S. cerevisiae .     

Lipid droplet autophagy in the yeast Saccharomyces cerevisiae

Cytosolic lipid droplets (LDs) are ubiquitous organelles in prokaryotes and eukaryotes that play a key role in cellular and organismal lipid homeostasis. Triacylglycerols (TAGs) and steryl esters, which are stored in LDs, are typically mobilized in growing cells or upon hormonal stimulation by LD-associated lipases and steryl ester hydrolases. Here we show that in the yeast Saccharomyces cerevisiae, LDs can also be turned over in vacuoles/lysosomes by a process that morphologically resembles microautophagy. A distinct set of proteins involved in LD autophagy is identified, which includes the core autophagic machinery but not Atg11 or Atg20. Thus LD autophagy is distinct from endoplasmic reticulum–autophagy, pexophagy, or mitophagy, despite the close association between these organelles. Atg15 is responsible for TAG breakdown in vacuoles and is required to support growth when de novo fatty acid synthesis is compromised. Furthermore, none of the core autophagy proteins, including Atg1 and Atg8, is required for LD formation in yeast.     

Ascospore formation in the yeast Saccharomyces cerevisiae.

Sporulation of the baker's yeast Saccharomyces cerevisiae is a response to nutrient depletion that allows a single diploid cell to give rise to four stress-resistant haploid spores. The formation of these spores requires a coordinated reorganization of cellular architecture. The construction of the spores can be broadly divided into two phases. The first is the generation of new membrane compartments within the cell cytoplasm that ultimately give rise to the spore plasma membranes. Proper assembly and growth of these membranes require modification of aspects of the constitutive secretory pathway and cytoskeleton by sporulation-specific functions. In the second phase, each immature spore becomes surrounded by a multilaminar spore wall that provides resistance to environmental stresses. This review focuses on our current understanding of the cellular rearrangements and the genes required in each of these phases to give rise to a wild-type spore.     

Mitochondrial iron metabolism in the yeast Saccharomyces cerevisiae

Iron is fundamental to many biological processes, but is also detrimental as it fosters the synthesis of destructive oxygen radicals. Recent experiments have increased our knowledge of the critical process of regulation of mitochondrial iron metabolism. A number of genes directly involved in iron homeostasis in this organelle have been identified. Intriguingly, a minor Hsp70 molecular chaperone of the mitochondrial matrix has been implicated as a player in this process as well.     

Atg9 trafficking in the yeast Saccharomyces cerevisiae

Autophagy can be divided into selective and nonselective modes. This process is considered selective when a precise cargo is specifically and exclusively incorporated into autophagosomes, the double-membrane vesicles that are the hallmark of autophagy. In contrast, during nonselective, bulk autophagy, cytoplasmic components are randomly enwrapped into autophagosomes. To date, approximately 30 autophagy-related genes called ATG have been identified. Sixteen of them compose the general basic machinery catalyzing the formation of double-membrane vesicles in all eukaryotic cells. The rest of them are often not conserved between species and cooperate with the basic Atg proteins during either selective or nonselective autophagy. Atg9 is the only integral membrane component of the conserved Atg machinery and appears to be a crucial organizational element. Recent studies in the S. cerevisiae have shown that Atg9 transport is differentially regulated depending on the autophagy mode. In this addendum, we will review and discuss what has recently been unveiled about yeast S. cerevisiae Atg9 trafficking, its modulators and its potential role in double-membrane vesicle biogenesis.