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E-GEOD-20351 - Transcription profiling by array of yeast PUN1 deletion mutants
Released on 17 February 2010, last updated on 30 April 2015
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 Σ1278b 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.
transcription profiling by array, genetic modification design
Anuj Kumar, Charles Boone, Christian A Shively, Craig J Dobry, Matthew J Eckwahl, Owen Ryan, Qingxuan Song, Rui Jin, Tao Xu
A profile of differentially abundant proteins at the yeast cell periphery during pseudohyphal growth. Xu T, Shively CA, Jin R, Eckwahl MJ, Dobry CJ, Song Q, Kumar A.