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SUB1 Plays a Negative Role during Starvation Induced Sporulation Program in Saccharomyces cerevisiae.

Gupta R, Sadhale PP, Vijayraghavan U - PLoS ONE (2015)

Bottom Line: Deletion of SUB1 gene significantly increased sporulation efficiency as compared to the wild-type cells in S288c genetic background.Deletion of SUB1 increased middle sporulation gene transcript levels with no effect on their induction kinetics.Taken together, our results suggest that SUB1 acts as a negative regulator of sporulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India.

ABSTRACT
Saccharomyces cerevisiae Sub1 is involved in several cellular processes such as, transcription initiation, elongation, mRNA processing and DNA repair. It has also been reported to provide cellular resistance during conditions of oxidative DNA damage and osmotic stress. Here, we report a novel role of SUB1 during starvation stress-induced sporulation, which leads to meiosis and spore formation in diploid yeast cells. Deletion of SUB1 gene significantly increased sporulation efficiency as compared to the wild-type cells in S288c genetic background. Whereas, the sporulation functions of the sub1(Y66A) missense mutant were similar to Sub1. SUB1 transcript and protein levels are downregulated during sporulation, in highly synchronized and sporulation proficient wild-type SK1 cells. The changes in Sub1 levels during sporulation cascade correlate with the induction of middle sporulation gene expression. Deletion of SUB1 increased middle sporulation gene transcript levels with no effect on their induction kinetics. In wild-type cells, Sub1 associates with chromatin at these loci in a temporal pattern that correlates with their enhanced gene expression seen in sub1Δ cells. We show that SUB1 genetically interacts with HOS2, which led us to speculate that Sub1 might function with Set3 repressor complex during sporulation. Positive Cofactor 4, human homolog of Sub1, complemented the sub1Δ sporulation phenotype, suggesting conservation of function. Taken together, our results suggest that SUB1 acts as a negative regulator of sporulation.

No MeSH data available.


Related in: MedlinePlus

Human PC4 can complements the yeast sub1Δ/sub1Δ sporulation phenotype.(A) Protein lysates were prepared from strains expressing either GFP or GFP-PC4 protein and were resolved by SDS-PAGE. Western blotting was done with anti GFP antibodies. (B) Cells expressing GFP-PC4 protein were analyzed by fluorescence microscopy to detect GFP or DAPI signals. (C) Wild type yeast SUB1 or Human PC4 was overexpressed from TEF2 promoter and 2μ plasmid in sub1Δ/sub1Δ strain. Sporulation was measured after 72 hours of transferring the cells to sporulation medium. Error bars represent standard deviation of three independent transformants for each strain (n = 1800) (NS, not significant).
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pone.0132350.g007: Human PC4 can complements the yeast sub1Δ/sub1Δ sporulation phenotype.(A) Protein lysates were prepared from strains expressing either GFP or GFP-PC4 protein and were resolved by SDS-PAGE. Western blotting was done with anti GFP antibodies. (B) Cells expressing GFP-PC4 protein were analyzed by fluorescence microscopy to detect GFP or DAPI signals. (C) Wild type yeast SUB1 or Human PC4 was overexpressed from TEF2 promoter and 2μ plasmid in sub1Δ/sub1Δ strain. Sporulation was measured after 72 hours of transferring the cells to sporulation medium. Error bars represent standard deviation of three independent transformants for each strain (n = 1800) (NS, not significant).

Mentions: S. cerevisiae Sub1 protein has a highly conserved N-terminal domain (1–107 aa), that has significant homology to even in its human counterpart PC4 [35] (S6 Fig). Besides this widely conserved domain is a C-terminal domain that is recognizable in its homologs from several other members of Saccharomycetaceae family like Candida glabrata, Vanderwaltozyma polyspora, Zygosaccharomyces rouxii and Ashbya gossypii (S6 Fig). The human PC4 (127 aa) and yeast Sub1 proteins (292 aa) share 48% identity and 70% similarity over the protein segment of 74 amino acids [1]. Therefore, we investigated whether human PC4 can complement the phenotypes of S. cerevisiae sub1Δ cells. We cloned the PC4 cDNA as N-terminal fusion with GFP in the yeast expression vector for expression from the TEF2 promoter. PC4 protein levels were clearly detected by Western blot analysis (Fig 7A) and immunofluoresence studies showed as in human cells, PC4 is nuclear localized in yeast cells too (Fig 7B). Overexpression of PC4 in sub1Δ/sub1Δ cells showed a reduction in sporulation efficiency with the extent of reduction being similar to that seen on overexpression of yeast full length SUB1. These experiments demonstrate that the human PC4 protein functionally complements the sub1Δ/sub1Δ mutant phenotype (Fig 7C).


SUB1 Plays a Negative Role during Starvation Induced Sporulation Program in Saccharomyces cerevisiae.

Gupta R, Sadhale PP, Vijayraghavan U - PLoS ONE (2015)

Human PC4 can complements the yeast sub1Δ/sub1Δ sporulation phenotype.(A) Protein lysates were prepared from strains expressing either GFP or GFP-PC4 protein and were resolved by SDS-PAGE. Western blotting was done with anti GFP antibodies. (B) Cells expressing GFP-PC4 protein were analyzed by fluorescence microscopy to detect GFP or DAPI signals. (C) Wild type yeast SUB1 or Human PC4 was overexpressed from TEF2 promoter and 2μ plasmid in sub1Δ/sub1Δ strain. Sporulation was measured after 72 hours of transferring the cells to sporulation medium. Error bars represent standard deviation of three independent transformants for each strain (n = 1800) (NS, not significant).
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Related In: Results  -  Collection

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pone.0132350.g007: Human PC4 can complements the yeast sub1Δ/sub1Δ sporulation phenotype.(A) Protein lysates were prepared from strains expressing either GFP or GFP-PC4 protein and were resolved by SDS-PAGE. Western blotting was done with anti GFP antibodies. (B) Cells expressing GFP-PC4 protein were analyzed by fluorescence microscopy to detect GFP or DAPI signals. (C) Wild type yeast SUB1 or Human PC4 was overexpressed from TEF2 promoter and 2μ plasmid in sub1Δ/sub1Δ strain. Sporulation was measured after 72 hours of transferring the cells to sporulation medium. Error bars represent standard deviation of three independent transformants for each strain (n = 1800) (NS, not significant).
Mentions: S. cerevisiae Sub1 protein has a highly conserved N-terminal domain (1–107 aa), that has significant homology to even in its human counterpart PC4 [35] (S6 Fig). Besides this widely conserved domain is a C-terminal domain that is recognizable in its homologs from several other members of Saccharomycetaceae family like Candida glabrata, Vanderwaltozyma polyspora, Zygosaccharomyces rouxii and Ashbya gossypii (S6 Fig). The human PC4 (127 aa) and yeast Sub1 proteins (292 aa) share 48% identity and 70% similarity over the protein segment of 74 amino acids [1]. Therefore, we investigated whether human PC4 can complement the phenotypes of S. cerevisiae sub1Δ cells. We cloned the PC4 cDNA as N-terminal fusion with GFP in the yeast expression vector for expression from the TEF2 promoter. PC4 protein levels were clearly detected by Western blot analysis (Fig 7A) and immunofluoresence studies showed as in human cells, PC4 is nuclear localized in yeast cells too (Fig 7B). Overexpression of PC4 in sub1Δ/sub1Δ cells showed a reduction in sporulation efficiency with the extent of reduction being similar to that seen on overexpression of yeast full length SUB1. These experiments demonstrate that the human PC4 protein functionally complements the sub1Δ/sub1Δ mutant phenotype (Fig 7C).

Bottom Line: Deletion of SUB1 gene significantly increased sporulation efficiency as compared to the wild-type cells in S288c genetic background.Deletion of SUB1 increased middle sporulation gene transcript levels with no effect on their induction kinetics.Taken together, our results suggest that SUB1 acts as a negative regulator of sporulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India.

ABSTRACT
Saccharomyces cerevisiae Sub1 is involved in several cellular processes such as, transcription initiation, elongation, mRNA processing and DNA repair. It has also been reported to provide cellular resistance during conditions of oxidative DNA damage and osmotic stress. Here, we report a novel role of SUB1 during starvation stress-induced sporulation, which leads to meiosis and spore formation in diploid yeast cells. Deletion of SUB1 gene significantly increased sporulation efficiency as compared to the wild-type cells in S288c genetic background. Whereas, the sporulation functions of the sub1(Y66A) missense mutant were similar to Sub1. SUB1 transcript and protein levels are downregulated during sporulation, in highly synchronized and sporulation proficient wild-type SK1 cells. The changes in Sub1 levels during sporulation cascade correlate with the induction of middle sporulation gene expression. Deletion of SUB1 increased middle sporulation gene transcript levels with no effect on their induction kinetics. In wild-type cells, Sub1 associates with chromatin at these loci in a temporal pattern that correlates with their enhanced gene expression seen in sub1Δ cells. We show that SUB1 genetically interacts with HOS2, which led us to speculate that Sub1 might function with Set3 repressor complex during sporulation. Positive Cofactor 4, human homolog of Sub1, complemented the sub1Δ sporulation phenotype, suggesting conservation of function. Taken together, our results suggest that SUB1 acts as a negative regulator of sporulation.

No MeSH data available.


Related in: MedlinePlus