A gene, SMP2, involved in plasmid maintenance and respiration in Saccharomyces cerevisiae encodes a highly charged protein.
ABSTRACT The smp2 mutant of Saccharomyces cerevisiae shows increased stability of the heterologous plasmid pSR1 and YRp plasmids. A DNA fragment bearing the SMP2 gene was cloned by its ability to complement the slow growth of the smp2 smp3 double mutant (smp3 is another mutation conferring increased stability of plasmid pSR1). The nucleotide sequence of SMP2 indicated that it encodes a highly charged 95 kDa protein. Disruption of the genomic SMP2 gene resulted in a respiration-deficient phenotype, although the cells retained mitochondrial DNA, and showed increased stability of pSR1 like the original smp2 mutant. The fact that the smp2 mutant is not always respiration deficient and shows increased pSR1 stability even in a rho0 strain lacking mitochondrial DNA suggested that the function of the Smp2 protein in plasmid maintenance is independent of respiration. The SMP2 locus was mapped at a site 71 cM from lys7 and 21 cM from ilv2/SMR1 on the right arm of chromosome XIII.
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ABSTRACT: Yeast PAH1-encoded phosphatidate phosphatase is the enzyme responsible for the production of the diacylglycerol used for the synthesis of triacylglycerol that accumulates in the stationary phase of growth. Paradoxically, the growth phase-mediated inductions of PAH1 and phosphatidate phosphatase activity do not correlate with the amount of Pah1p; enzyme abundance declined in a growth phase-dependent manner. Pah1p from exponential phase cells was a relatively stable protein and its abundance was not affected by incubation with an extract from stationary phase cells. Recombinant Pah1p was degraded upon incubation with the 100,000 x g pellet fraction of stationary phase cells, whereas, the enzyme was stable when incubated with the same fraction of exponential phase cells. MG132, an inhibitor of proteasome function, prevented degradation of the recombinant enzyme. Endogenously expressed and plasmid-mediated overexpressed levels of Pah1p were more abundant in the stationary phase of cells treated with MG132. Pah1p was stabilized in mutants with impaired proteasome (rpn4Δ, blm10Δ, ump1Δ, and pre1 pre2 mutations) and ubiquitination (hrd1Δ, ubc4Δ, ubc7Δ, ubc8Δ, and doa4Δ) functions. The pre1 pre2 mutations that eliminate nearly all chymotrypsin-like activity of the 20S proteasome had the greatest stabilizing effect on enzyme levels. Taken together, these results supported the conclusion that Pah1p is subject to proteasome-mediated degradation in the stationary phase. That Pah1p abundance was stabilized in pah1Δ mutant cells expressing catalytically inactive forms of Pah1p and dgk1Δ mutant cells with induced expression of DGK1-encoded diacylglycerol kinase indicated that alteration in phosphatidate and/or diacylglycerol levels might be the signal that triggers Pah1p degradation.Journal of Biological Chemistry 02/2014; DOI:10.1074/jbc.M114.550103 · 4.60 Impact Factor
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ABSTRACT: Yeast Pah1p is the phosphatidate phosphatase that catalyzes the penultimate step in triacylglycerol synthesis and plays a role in the transcriptional regulation of phospholipid synthesis genes. The enzyme is multiply phosphorylated, some of which is mediated by Pho85p-Pho80p, Cdc28p-cyclin B, and protein kinase A. Here, we showed that Pah1p is a bona fide substrate of protein kinase C; the phosphorylation reaction was time- and dose-dependent, and dependent on the concentrations of ATP (Km = 4.5 µM) and Pah1p (Km = 0.75 µM). The stoichiometry of the reaction was 0.8 mol phosphate/mol Pah1p. By combining mass spectrometry, truncation analysis, site-directed mutagenesis, and phosphopeptide mapping, we identified Ser-677, Ser-769, Ser-773, and Ser-788 as major sites of phosphorylation. Analysis of Pah1p phosphorylations by different protein kinases showed that pre-phosphorylation with protein kinase C reduced its subsequent phosphorylation with protein kinase A, and vice versa. Pre-phosphorylation with Pho85p-Pho80p had an inhibitory effect on its subsequent phosphorylation with protein kinase C; however, pre-phosphorylation with protein kinase C had no effect on the subsequent phosphorylation with Pho85p-Pho80p. Unlike its phosphorylations by Pho85p-Pho80p and protein kinase A, which cause a significant reduction in phosphatidate phosphatase activity, the phosphorylation of Pah1p by protein kinase C had a small stimulatory effect on the enzyme activity. Analysis of phosphorylation-deficient forms of Pah1p indicated that protein kinase C does not have a major effect on its location or its function in triacylglycerol synthesis, but instead, the phosphorylation favors loss of Pah1p abundance when it is not phosphorylated with Pho85p-Pho80p.Journal of Biological Chemistry 05/2014; DOI:10.1074/jbc.M114.581462 · 4.60 Impact Factor
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ABSTRACT: In the yeast Saccharomyces cerevisiae, the synthesis of phospholipids in the exponential phase of growth occurs at the expense of the storage lipid triacylglycerol. As exponential phase cells progress into the stationary phase, the synthesis of triacylglycerol occurs at the expense of phospholipids. Early work indicates a role of the phosphatidate phosphatase (PAP) in this metabolism; the enzyme produces the diacylglycerol needed for the synthesis of triacylglycerol and simultaneously controls the level of phosphatidate for the synthesis of phospholipids. Four genes (APP1, DPP1, LPP1, and PAH1) encode PAP activity in yeast, and it has been unclear which gene is responsible for the synthesis of triacylglycerol throughout growth. An analysis of lipid synthesis and composition, as well as PAP activity in various PAP mutant strains showed the essential role of PAH1 in triacylglycerol synthesis throughout growth. Pah1p is a phosphorylated enzyme whose in vivo function is dependent on its dephosphorylation by the Nem1p-Spo7p protein phosphatase complex. nem1Δ mutant cells exhibited defects in triacylglycerol synthesis and lipid metabolism that mirrored those imparted by the pah1Δ mutation, substantiating the importance of Pah1p dephosphorylation throughout growth. An analysis of cells bearing PPAH1-lacZ and PPAH1-DPP1 reporter genes showed that PAH1 expression was induced throughout growth and that the induction in the stationary phase was stimulated by inositol supplementation. A mutant analysis indicated that the Ino2p/Ino4p,/Opi1p regulatory circuit and transcription factors Gis1p and Rph1p mediated this regulation.Journal of Biological Chemistry 11/2013; DOI:10.1074/jbc.M113.525766 · 4.60 Impact Factor