Human OVCA2 and its homolog FSH3-induced apoptosis in Saccharomyces cerevisiae.

Mammalian ovarian tumor suppressor candidate 2 (OVCA2) gene belongs to the family of serine hydrolase (FSH). This study aimed to elucidate the functional similarities of OVCA2 with its yeast homolog genes (FSH1, FSH2, and FSH3) regarding apoptosis. We found that the expression of OVCA2 in Saccharomyces cerevisiae increased production of reactive oxygen species (ROS), decreased cell growth, disturbed mitochondrial morphology, reduced membrane potential, increased chromatin condensation, and externalization of phosphatidylserine (PS) (annexin V/propidium iodide staining) indicating induced apoptotic cell death in yeast. We also showed that complementation of OVCA2 in fsh3Δ cells reduced cell growth and increased the apoptotic phenotypes. Collectively, our results suggest that complementation of human OVCA2 in fsh3Δ cells induced apoptosis in S. cerevisiae.

FSH1 overexpression triggers apoptosis in Saccharomyces cerevisiae.

FSH1 belongs to the family of serine hydrolases in yeast and is homologous to the human ovarian tumor suppressor gene (OVAC2). Our preliminary results showed that cells lacking Fsh1p exhibit an increase in cell growth, and a decrease in the expression of AIF1 and NUC1 (apoptosis responsive genes) when compared to the wild type cells. Growth inhibition of cells overexpressing FSH1 is due to induction of cell death associated with cell death markers typical of mammalian apoptosis namely DNA fragmentation, phosphatidylserine externalization, ROS accumulation, Cytochrome c release, and altered mitochondrial membrane potential. When wild type cells were overexpressed with FSH1 there was up regulation of AIF1 level when compared to control cells suggesting that overexpression of FSH1 regulated cell death in yeast.

Impaired GCR1 transcription resulted in defective inositol levels, vacuolar structure and autophagy in Saccharomyces cerevisiae.

In yeast, the GCR1 transcription factor is involved in the regulation of glycolysis and its deletion exhibited growth defect, reduced inositol and phosphatidylinositol (PI) levels compared to WT cells. We observed a down regulation of the INO1 and PIS1 expression in gcr1∆ cells under both I- and I+ conditions and the over expression of GCR1 in gcr1∆ cells restored the growth, retrieved the expression of INO1, and PIS1 comparable to WT cells. In the gel shift assay, the Gcr1p binds to its consensus sequence CTTCC in PIS1 promoter and regulates its expression but not in INO1 transcription. The WT cells, under I- significantly reduced the expression of GCR1 and PIS1, but increased the expression of KCS1 and de-repressed INO1. The Kcs1p expression was reduced in gcr1∆ cells; this reduced INO1 expression resulting in abnormal vacuolar structure and reduced autophagy in Saccharomyces cerevisiae.

FSH3 mediated cell death is dependent on NUC1 in Saccharomyces cerevisiae.

Family of Serine Hydrolases (FSH) members FSH1, FSH2 and FSH3 in Saccharomyces cerevisiae share conserved sequences with the human candidate tumor suppressor OVCA2. In this study, hydrogen peroxide (H2O2) exposure increased the expression of both mRNA and protein levels of FSH3 in wild-type (WT) yeast cells. The deletion of FSH3 improved the yeast growth rate under H2O2-induction as compared to WT control cells. The overexpression of FSH3 in WT yeast cells caused an apoptotic phenotype, including accumulation of reaction oxygen species, decreased cell viability and cell death. The double deletions fsh1Δ fsh2Δ, fsh1Δ fsh3Δ and fsh2Δ fsh3Δ displayed increased growth compared to WT cells. However, the overexpression of FSH3 effectively inhibited cell growth in all double deletions. Moreover, the overexpression of FSH3 in cells lacking NUC1 did not cause any growth defect in the presence or absence of H2O2. Our results suggest that FSH3 induced apoptosis of yeast in a NUC1 dependent manner.

Dolichyl pyrophosphate phosphatase-mediated N-glycosylation defect dysregulates lipid homeostasis in Saccharomyces cerevisiae.

The endoplasmic reticulum (ER) has numerous biological functions including protein synthesis, protein folding, and lipid synthesis. The CAX4 gene encodes dolichyl pyrophosphate (Dol-PP) phosphatase, which is involved in protein N-glycosylation. In cax4Δ cells, the N-glycosylation of the vacuolar carboxypeptidase (CPY) was severely affected, and expression of the ER chaperone Kar2p was elevated, which resulted in UPR activation as an adaptive response. The cax4Δ cell growth was reduced, and this could be attributed to the formation of clumped aggregates, high vesiculation of the intracellular membrane, and plasma membrane alterations were depicted using DiOC6 fluorescence. In the cax4 deletion strain, the transcription factors INO2 and INO4 were upregulated, and the negative regulator OPI1 was concomitantly down regulated, which led to the derepression of the phospholipid genes CHO2, OPI3, PSD1, and PSD2 and resulted in increased phospholipid levels. However, the TAG, SE, and LD levels were significantly reduced, and FFA, sterol, and DAG levels were increased. These findings could be attributed to the derepression of the TAG and SE lipases TGL3, TGL4, TGL5, YEH1, and YEH2 and the repression of LRO1, DGA1, ARE1, and ARE2 in cax4Δ cells. Interestingly, the overexpression of SEC59 or CAX4 in cax4Δ cells prevented the ER stress and growth defect, and restored normal level of phospholipids, neutral lipids, and LDs. The current study revealed the disruption of N-glycosylation-induced ER stress, altered lipid homeostasis accounts for pleiotropic phenotype. Thus, CAX4 regulates membrane biogenesis by coordinating lipid homeostasis with protein quality control.

MGL2/YMR210w encodes a monoacylglycerol lipase in Saccharomyces cerevisiae.

In silico analysis of the uncharacterized open reading frame YMR210w in Saccharomyces cerevisiae revealed that it possesses both an α/ß hydrolase domain (ABHD) and a typical lipase (GXSXG) motif. The purified protein displayed monoacylglycerol (MAG) lipase activity and preferred palmitoyl-MAG. Overexpression of YMR210w in the known MAG lipase mutant yju3Δ clearly revealed that the protein had MAG lipase activity, hence we named the ORF MGL2. Overexpression of YMR210w decreased the cellular triacylglycerol levels. Analysis of the overexpressed strains showed reduction in the lipid droplets number and size. Phenotype studies revealed that the double deletion yju3Δmgl2Δ displayed a growth defect that was partially restored by MGL2 overexpression.