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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

sub1(Y66A) missense mutant is competent for sporulation functions.Wild type SUB1 or sub1(Y66A) mutant was over-expressed from TEF2 promoter and 2μ plasmid in S288c 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 = 2000) (NS, not significant).
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pone.0132350.g002: sub1(Y66A) missense mutant is competent for sporulation functions.Wild type SUB1 or sub1(Y66A) mutant was over-expressed from TEF2 promoter and 2μ plasmid in S288c 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 = 2000) (NS, not significant).

Mentions: Previously, it has been reported that Sub1 has a role in transcriptional repression of IMP dehydrogenase 2 (IMD2) [25]. Moreover, it is suggested that Sub1 and replication factor A (Rfa1) compete with each other for binding to ssDNA in transcription complexes. This conclusion was based on increased occupancy of Rfa1 at the promoter regions of several actively transcribed genes in haploid cells lacking Sub1 [5]. Interestingly the same study showed that sub1(Y66A) missense mutant showed increased IMD2 gene expression with occupancy of Rfa1 at active promoters in the sub1(Y66A) missense mutant being similar to that in sub1Δ strain [5]. Because sub1(Y66A) missense mutant displayed phenotypes similar to the loss of function allele, in the haploid cells, we investigated the role of sub1(Y66A) mutant in sporulation response of diploid cells subjected to severe starvation. Towards this aim, we assessed sporulation efficiency of sub1Δ/sub1Δ S288c cells transformed with plasmid over-expressing either sub1(Y66A) mutant allele or the wild-type SUB1. Expression of the full length Sub1 protein reduced the sporulation efficiency to near S288c wild-type levels (Fig 2). Interestingly, the over-expression of sub1(Y66A) mutant in strain lacking endogenous Sub1 also showed a reduction in sporulation efficiency to levels achieved by the plasmid expressed wild type Sub1. This result suggests that the sub1(Y66A) missense mutant is competent for the sporulation functions of Sub1. To ascertain comparable protein levels, Western blot analysis was performed on protein lysates from sub1Δ/sub1Δ cells expressing GFP fusions with either wild-type SUB1 or sub1(Y66A) missense mutant (S2A Fig). As we detect equivalent levels of Sub1(Y66A) and wild-type Sub1 proteins, we conclude that the missense mutation does not alter protein stability. Genome-wide immunolocalization study of yeast proteins in haploid log-phase cells reported Sub1 to be a nuclear protein [26]. We validated the nuclear localization of wild-type GFP-Sub1 and found that in haploid log-phase cells the GFP-Sub1(Y66A) mutant protein is also nuclear localized (S2B Fig). While functional for repression of sporulation in diploid cells, the sub1(Y66A) mutant does not repress IMD2 gene expression, as the transcript levels of IMD2 gene in sub1(Y66A) mutant were comparable to sub1Δ deletion strain (S2C Fig), in accord with a previous report of its effects on IMD2 in haploid vegetative cells [5]. Taken together, our result indicates that sub1(Y66A) is competent for sporulation functions.


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

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

sub1(Y66A) missense mutant is competent for sporulation functions.Wild type SUB1 or sub1(Y66A) mutant was over-expressed from TEF2 promoter and 2μ plasmid in S288c 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 = 2000) (NS, not significant).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4492983&req=5

pone.0132350.g002: sub1(Y66A) missense mutant is competent for sporulation functions.Wild type SUB1 or sub1(Y66A) mutant was over-expressed from TEF2 promoter and 2μ plasmid in S288c 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 = 2000) (NS, not significant).
Mentions: Previously, it has been reported that Sub1 has a role in transcriptional repression of IMP dehydrogenase 2 (IMD2) [25]. Moreover, it is suggested that Sub1 and replication factor A (Rfa1) compete with each other for binding to ssDNA in transcription complexes. This conclusion was based on increased occupancy of Rfa1 at the promoter regions of several actively transcribed genes in haploid cells lacking Sub1 [5]. Interestingly the same study showed that sub1(Y66A) missense mutant showed increased IMD2 gene expression with occupancy of Rfa1 at active promoters in the sub1(Y66A) missense mutant being similar to that in sub1Δ strain [5]. Because sub1(Y66A) missense mutant displayed phenotypes similar to the loss of function allele, in the haploid cells, we investigated the role of sub1(Y66A) mutant in sporulation response of diploid cells subjected to severe starvation. Towards this aim, we assessed sporulation efficiency of sub1Δ/sub1Δ S288c cells transformed with plasmid over-expressing either sub1(Y66A) mutant allele or the wild-type SUB1. Expression of the full length Sub1 protein reduced the sporulation efficiency to near S288c wild-type levels (Fig 2). Interestingly, the over-expression of sub1(Y66A) mutant in strain lacking endogenous Sub1 also showed a reduction in sporulation efficiency to levels achieved by the plasmid expressed wild type Sub1. This result suggests that the sub1(Y66A) missense mutant is competent for the sporulation functions of Sub1. To ascertain comparable protein levels, Western blot analysis was performed on protein lysates from sub1Δ/sub1Δ cells expressing GFP fusions with either wild-type SUB1 or sub1(Y66A) missense mutant (S2A Fig). As we detect equivalent levels of Sub1(Y66A) and wild-type Sub1 proteins, we conclude that the missense mutation does not alter protein stability. Genome-wide immunolocalization study of yeast proteins in haploid log-phase cells reported Sub1 to be a nuclear protein [26]. We validated the nuclear localization of wild-type GFP-Sub1 and found that in haploid log-phase cells the GFP-Sub1(Y66A) mutant protein is also nuclear localized (S2B Fig). While functional for repression of sporulation in diploid cells, the sub1(Y66A) mutant does not repress IMD2 gene expression, as the transcript levels of IMD2 gene in sub1(Y66A) mutant were comparable to sub1Δ deletion strain (S2C Fig), in accord with a previous report of its effects on IMD2 in haploid vegetative cells [5]. Taken together, our result indicates that sub1(Y66A) is competent for sporulation functions.

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