SEC6 encodes an 85 kDa soluble protein required for exocytosis in yeast.

The SEC6 gene encodes a protein required for an event leading to fusion of post-Golgi vesicles with the plasma membrane in Saccharomyces cerevisiae cells. The gene was cloned by complementation of the temperature-sensitive growth defect of a sec6-4 strain. The nucleotide sequence was determined and the longest open reading frame was found to encode an 85 kDa protein of 733 amino acids. The Sec6 protein is predicted to be hydrophilic and is found predominantly in the soluble fraction of a yeast lysate, in a species that sediments with a coefficient of 14S. No extensive homology was found with known proteins of the database. Gene disruption and marker rescue experiments indicate that SEC6 is a single copy gene essential for growth. Overproduction of Sec6p does not suppress any of the other late-acting sec mutants, yet sec6-4 does display synthetic lethality with sec8-9, suggesting that the two gene products may fulfill inter-related functions.     

The plasma membrane H+-ATPase,a simple polypeptide with a long history

The plasma membrane H⁺-ATPase of fungi and plants is a single polypeptide of fewer than 1,000 residues that extrudes protons from the cell against a large electric and concentration gradient. The minimalist structure of this nanomachine is in stark contrast to that of the large multi-subunit F_OF₁ ATPase of mitochondria, which is also a proton pump, but under physiological conditions runs in the reverse direction to act as an ATP synthase. The plasma membrane H⁺-ATPase is a P-type ATPase, defined by having an obligatory phosphorylated reaction cycle intermediate, like cation pumps of animal membranes, and thus, this pump has a completely different mechanism to that of F_OF₁ ATPases, which operates by rotary catalysis. The work that led to these insights in plasma membrane H⁺-ATPases of fungi and plants has a long history, which is briefly summarized in this review.     

GSG1, a yeast gene required for sporulation

We have identified a gene, GSG1 (g eneral s porulation g ene 1), required for sporulation in Saccharomyces cerevisiae. Diploids homozygous for a disruption of GSG1 fail to sporulate. The gene has an open reading frame of 2094 bp, encoding a polypeptide with an expected size of 77 kDa. GSG1 is expressed mitotically in both a and α haploids, and both mitotically and meiotically in diploids. The message level of GSG1 increases approximately two-fold after 4–6 h of sporulation. gsg1 mutants enter pre-meiotic DNA synthesis later than wild-type diploids. Mutant diploids are not rescued by spo13. These results suggest that GSG1 has a role late in meiosis following DNA replication. The sequence reported here has the GenBank Accession Number U26674.     

A genomic sequence of the Schizosaccharomyces pombe 16 kDa vacuolar H(+)-ATPase.

We have isolated the gene encoding the 16 kDa vacuolar H(+)-ATPase from Schizosaccharomyces pombe. On the basis of RNA splicing signals and amino acid sequence homology with other 16 kDa H(+)-ATPases, the genomic DNA sequence indicated the 16 kDa protein is encoded by five exons. The C-terminal 50 amino acids has more than 90% homology with vacuolar H(+)-ATPases of mammalian cells.     

Sequence,mapping and disruption of CCC2, a gene that cross-complements the Ca2+-sensitive phenotype of csg1 mutants and encodes a P-type ATPase belonging to the Cu2+-ATPase subfamily

We have isolated, sequenced, mapped and disrupted a gene, CCC2, from Saccharomyces cerevisiae. This gene displays non-allelic complementation of the Ca²⁺-sensitive phenotype conferred by the csg1 mutation. Analysis of the CCC2p amino acid sequence reveals that it encodes a member of the P-type ATPase family and is most similar to a subfamily thought to consist of Cu²⁺ transporters, including the human genes that mutate to cause Wilson disease and Menkes disease. The ability of this gene, in two or more copies, to reverse the csg1 defect suggests that Ca²⁺-induced death of csg1 mutant cells is related to Cu²⁺ metabolism. Cells without CCC2 require increased Cu²⁺ concentrations for growth. Therefore CCC2p may function to provide Cu²⁺ to a cellular compartment rather than in removal of excess of Cu²⁺. The sequence of CCC2 is available through GenBank under accession number L36317.     

The Saccharomyces cerevisiae TGL2 gene encodes a protein with lipolytic activity and can complement an Escherichia coli diacylglycerol kinase disruptant

Escherichia coli cells with a disrupted diacylglycerol kinase gene are unable to grow on media containing arbutin due to a lethal accumulation of diacylglycerol. In order to isolate genes from the yeast Saccharomyces cerevisiae involved in diacylglycerol metabolism we complemented an E. coli diacylglycerol kinase disruptant with a yeast genomic library and transformants were selected capable of growing in the presence of arbutin. Using this method, a gene (TGL2) was isolated coding for a protein resembling lipases from Pseudomonas. After expression of the TGL2 gene in E. coli, lipolytic activity towards triacylglycerols and diacylglycerols with short-chain fatty acids could be measured. Therefore, it is very likely that the TGL2 gene can complement the E. coli diacylglycerol kinase disruptant, because it encodes a protein that degrades the diacylglycerol accumulated after growth in the presence of arbutin. Disruption of the TGL2 gene in S. cerevisiae did not result in a detectable phenotype. The role of the Tgl2 protein in lipid degradation in yeast is still unclear. The nucleotide sequence published here has been submitted to the EMBL sequence data bank and is available under accession number X98000. © 1998 John Wiley & Sons, Ltd.     

Sequence of the genes encoding subunits A and B of the vacuolar H(+)-ATPase of Schizosaccharomyces pombe.

The genes encoding subunits A (vma1) and B (vma2) of the vacuolar H(+)-ATPase from Schizosaccharomyces pombe were cloned by hybridization to cDNAs of the homologous genes in Neurospora crassa. Both genes are interrupted by introns, two in vma1 and four in vma2. Positions of introns do not appear to be conserved when compared to those of N. crassa. The subunit A gene encodes a single product of 619 amino acids and is not interrupted by the coding sequence for a second product as found for Saccharomyces cerevisiae (Kane, P. K., Yamashiro, C. T., Wolczyk, D. F., Neff, N., Goebl, M., and Stevens, T. H. (1990). Science 250, 651-657).     

Characterization of vps33+, a gene required for vacuolar biogenesis and protein sorting in Schizosaccharomyces pombe

From the fission yeast Schizosaccharomyces pombe we have identified and deleted vps33, a gene encoding a homologue of VPS33, which is required for vacuolar biogenesis in S. cerevisiae cells. When the vps33(+) gene is disrupted, Sz. pombe strains are temperature-sensitive for growth and contain numerous small vesicular structures stained with FM4-64 in the cells. Deletion of the Sz. pombe vps33(+) gene results in pleiotropic phenotypes consistent with the absence of normal vacuoles, including missorting of vacuolar carboxypeptidase Y, various ion- and drug-sensitivities, and sporulation defects. These results are consistent with Vps33p being necessary for the morphogenesis of vacuoles and subsequent expression of vacuolar functions in Sz. pombe cells.     

The yeast rRNA biosynthesis factor Ebp2p is also required for efficient nuclear division

Molecular genetic analysis of the yeast Ebp2 protein has revealed that it is an essential, nucleolar protein that functions in the rRNA biosynthesis pathway. Temperature-sensitive ebp2-1 mutants are defective in the processing of the 27 SA precursor rRNA, and the point substitutions that disrupt this activity cluster towards the central, more highly conserved region of the Ebp2 protein. We report here that other ebp2 mutants exhibit deficiencies associated with defects in chromosome segregation. Yeast cells bearing a 50 amino acid C-terminal truncation allele (ebp2 delta C50) display a slow-growth phenotype and exhibit an increased percentage of cells with the nucleus positioned at the bud neck. The ebp2-1 and ebp2 delta C50 alleles genetically complement each other, and ebp2 delta C50 mutants exhibit nuclear division defects that are distinct from the rRNA biosynthesis-related phenotypes of ebp2-1 mutants. Cytological and FACS analysis of the ebp2 delta C50 deletion mutants indicate that the chromosome segregation related activities of the Ebp2 protein are monitored by Mad2p, a mitotic checkpoint protein. The finding that yeast Ebp2p functions in nuclear division is consistent with the growing body of evidence that supports the role that human EBP2 plays in chromosome segregation.     

The IRC7 gene encodes cysteine desulphydrase activity and confers on yeast the ability to grow on cysteine as a nitrogen source

Although cysteine desulphydrase activity has been purified and characterized from Saccharomyces cerevisiae, the gene encoding this activity in vivo has never been defined. We show that the full‐length IRC7 gene, encoded by the YFR055W open reading frame, encodes a protein with cysteine desulphydrase activity. Irc7p purified to homogeneity is able to utilize l ‐cysteine as a substrate, producing pyruvate and hydrogen sulphide as products of the reaction. Purified Irc7p also utilized l ‐cystine and some other cysteine conjugates, but not l ‐cystathionine or l ‐methionine, as substrates. We further show that, in vivo, the IRC7 gene is both necessary and sufficient for yeast to grow on l ‐cysteine as a nitrogen source, and that overexpression of the gene results in increased H₂S production. Strains overexpressing IRC7 are also hypersensitive to a toxic analogue, S‐ethyl‐l ‐cysteine. While IRC7 has been identified as playing a critical role in converting cysteine conjugates to volatile thiols that are important in wine aroma, its biological role in yeast cells is likely to involve regulation of cysteine and redox homeostasis. Copyright © 2015 John Wiley & Sons, Ltd.     

The in vivo activation of Saccharomyces cerevisiae plasma membrane H+-ATPase by ethanol depends on the expression of the PMA1 gene,but not of the PMA2 gene

The expression of the PMA1 and PMA2 genes during Saccharomyces cerevisiae growth in medium with glucose plus increasing concentrations of ethanol was monitored by using PMA1-lacZ and PMA2-lacZ fusions and Northern blot hybridizations of total RNA probed with PMA1 gene. The presence of sub-lethal concentrations of ethanol enhanced the expression of PMA2 whereas it reduced the expression of PMA1. The inhibition of PMA1 expression by ethanol corresponded to a decrease in the content of plasma membrane ATPase as quantified by immunoassays. Although an apparent correspondence could exist between the increase of plasma membrane ATPase activity and the level of PMA2 expression, the maximal level of PMA2 expression remained about 200 times lower than PMA1. On the other hand, ethanol activated the plasma membrane H⁺-ATPase activity from a strain expressing only the PMA1 ATPase but did not activate that from a strain expressing only the PMA2 ATPase. These results provide evidence that in the presence of ethanol it is the PMA1 ATPase which is activated, probably by a post-translational mechanism and that the PMA2 ATPase is not involved.     

Molecular characterization of a gene that confers 2-deoxyglucose resistance in yeast

We have isolated a gene whose expression enables yeast cells to overcome the inhibition of growth produced by the presence of 2-deoxyglucose. The gene contains an open reading frame of 738 bp that may code for a protein of 27 100 Da. Cells carrying this gene contain high levels of a specific 2-deoxyglucose-6-phosphate phosphatase. The expression of this phosphatase is increased by the presence of 2-deoxyglucose and is constant along the growth curve. The sequence reported here has the GenBank accession number U03107.     

The activity of plasma membrane H(+)-ATPase is strongly stimulated during Saccharomyces cerevisiae adaptation to growth under high copper stress, accompanying intracellular acidification

For the adaptation of cells of Saccharomyces cerevisiae, a period of latency is necessary before exponential growth is resumed in a medium supplemented with a highly inhibitory concentration of copper. In this work, we have examined some physiological responses occurring during this period of adaptation. The results revealed that plasma membrane H(+)-ATPase (PM-ATPase) activity is strongly stimulated (up to 24-fold) during copper-induced latency in growth medium with glucose, reaching maximal levels when the cells were about to start inhibited exponential growth. This in vivo activation of the ATPase activity by copper was accompanied by the stimulation of the H(+)-pumping activity of the enzyme in vivo and was essentially due to the increase of the apparent V(max) for MgATP. Although the exact molecular basis of the reported plasma membrane ATPase activation was not clarified, no increase in the mRNA levels from the encoding genes PMA1 and PMA2 was apparently detected during copper-induced latency. The physiological response reported here may allow the cells to cope with copper-induced lipid peroxidation and consequent decrease in plasma membrane lipid ordering and increase in the non-specific permeability to protons. The consequences of these copper deleterious effects were revealed by the decrease of the intracellular pH (pH(i)) of the yeast population, from approximately pH(i) 6 to pH(i) 5, during copper-induced latency in growth medium at pH 4.3. The time-dependent patterns of plasma membrane ATPase activation and of the decrease of pH(i) during the period of adaptation to growth with copper correlate, suggesting that the regulation of this membrane enzyme activity may be triggered by intracellular acidification. Consistent with this idea, when exponential growth under copper stress was resumed and the pH(i) of the yeast population recovered up to physiological values, plasma membrane ATPase activity simultaneously decreased from the highly stimulated level attained during the adaptation period of latency.     

Localization of a protein a-tagged kex2 protein to the vacuole of Saccharomyces cerevisiae allows rapid purification of vacuolar membranes

We have previously reported an immunoisolation procedure which allows purification of Kex2p-containing Golgi membranes from lysed yeast cells. In order to evaluate the use of tagging procedures in organelle isolation we set out to isolate the same Golgi membrane fraction using a version of the Kex2 protease that had been affinity-tagged at its C-terminus. This protein is found to be localized in the vacuole, providing the basis of a method for the affinity-purification of vacuolar membranes.     

The sequence of a 17.5 kb DNA fragment on the left arm of yeast chromosome XI identifies the protein kinase gene ELM1, the DNA primase gene PRI2, a new gene encoding a putative histone and seven new open reading frames

A 17.5 kb DNA fragment of chromosome XI, located between the genetic loci mif2 and mak11 was sequenced and analysed. Ten open reading frames were identified. Two of them are the previously sequenced genes ELM1 and PRI2, two (YKL253 and YKL256) show homologies to proteins from other organisms and one (YKL262) to yeast and mouse histone.