Unknown,Transcriptomics,Genomics,Proteomics

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Yeast_FLO8_MSS11_Deletions_LowN

ABSTRACT: In filamentous strains of S. cerevisiae, nitrogen stress elicits a complex morphogenetic program resulting in the transition to a filamentous form of growth. The transcription factors Flo8p and Mss11p are both required for this filamentous growth transition, in that homozygous diploid flo8(delta/delta) and mss11(delta/delta) strains do not undergo filamentation under conditions of nitrogen stress. To identify genes that are regulated either directly or indirectly by the Flo8p and Mss11p transcription factors, we have implemented DNA microarray analysis to profile changes in mRNA levels in homozygous diploid strains of the filamentous (Sigma)1278b genetic background deleted for FLO8 and MSS11, respectively, under conditions of nitrogen stress. The transcriptional profiles will identify genes with altered transcriptional levels in the deletion mutants, serving as a means to identify cellular processes and signaling pathways regulated by Flo8p and Mss11p function. Deletion mutants were constructed using a one-step polymerase chain reaction (PCR)-based gene disruption strategy with the G418 resistance cassette from plasmid pFA6a-KanMX6. After the haploid mutants were confirmed via PCR, the strains were allowed to mate on YPD+G418 plates for approximately 20 hours at 30 degrees Celsius. Mated cells were then streaked on SC-Trp-Leu plates to select for Y825 x HLY337 diploids. Yeast transformations were performed according to standard lithium acetate-mediated protocols with modifications. Yeast strains (wild-type and deletion mutants) were cultured under conditions of nitrogen stress (low ammonium media), and RNA was prepared using the Poly(A) Purist kit (Ambion, Austin, TX). RNA concentration and purity were determined spectrophotometrically and by gel electrophoresis. Microarray hybridization was performed with the Yeast Genome S98 Array using standard protocols (Affymetrix, Inc, Santa Clara, CA). Differentially expressed genes were identified by significance analysis of microarrays (SAM). SAM computed a nonparametric score for each gene by dividing the between-group difference of (normalized log) gene expression levels and the within-group difference of gene expression levels. The score was then compared with random permutation scores. The random permutation scores for a gene were computed in the same manner as the original score, but based on randomly sampled gene expressions. If the difference between the original score and the random permutation score was larger than a chosen threshold value, the corresponding change in gene expression was claimed to be significant. Each threshold value corresponded to a false discovery rate (the percentage of genes identified as being significant by chance alone). For all deletion comparisons in this study, we used SAM’s two-class unpaired analysis function, with significance thresholds selected so that the corresponding false discovery rate was 0.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Anuj Kumar

PROVIDER: E-GEOD-40530 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Yeast filamentous growth is a stress response to conditions of nitrogen deprivation, wherein yeast colonies form pseudohyphal filaments of elongated and connected cells. As proteins mediating adhesion and transport are required for this growth transition, the protein complement at the yeast cell periphery plays a critical and tightly regulated role in enabling pseudohyphal filamentation. To identify proteins differentially abundant at the yeast cell periphery during pseudohyphal growth, we generated quantitative proteomic profiles of plasma membrane protein preparations under conditions of vegetative growth and filamentation. By iTRAQ chemistry and two-dimensional LC-MS/MS, we profiled 2,463 peptides and 356 proteins, from which we identified eleven differentially abundant proteins that localize to the yeast cell periphery. This protein set includes Ylr414cp, herein renamed Pun1p, a previously uncharacterized protein localized to the plasma membrane compartment of Can1 (MCC). Pun1p abundance is increased two-fold under conditions of nitrogen stress, and deletion of PUN1 abolishes filamentous growth in haploids and diploids; pun1D mutants are non-invasive, lack surface-spread filamentation, grow slowly, and exhibit impaired cell adhesion. Conversely, overexpression of PUN1 results in exaggerated cell elongation under conditions of nitrogen stress. PUN1 contributes to yeast nitrogen signaling, as pun1D mutants misregulate amino acid biosynthetic genes during nitrogen deprivation. By chromatin immunoprecipitation and RT-PCR, we find that the filamentous growth factor Mss11p directly binds to the PUN1 promoter and regulates its transcription. In total, this study provides the first profile of protein abundance during pseudohyphal growth, identifying a previously uncharacterized MCC protein required for wild-type nitrogen signaling and filamentous growth. For this study, we constructed a homozygous diploid strain in the filamentous ?1278b background deleted for PUN1; a wild-type diploid strain of the same background served as the control. Both strains were grown under conditions of low nitrogen, and RNA was extracted from three biological replicates of each strain after identical culturing. The RNA samples were analyzed using affymerix DNA microarrays.

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Yeast_FLO8_MSS11_Deletions_LowN

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