A Profile of Differentially Abundant Proteins at the Yeast Cell Periphery during Pseudohyphal Growth

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, we expect that the protein complement at the yeast cell periphery plays a critical and tightly regulated role in 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 isobaric tags for relative and absolute quantification chemistry and two-dimensional liquid chromatography-tandem mass spectrometry, we profiled 2463 peptides and 356 proteins, identifying 11 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. Pun1p abundance is doubled under conditions of nitrogen stress, and deletion of PUN1 abolishes filamentous growth in haploids and diploids; pun1 Delta mutants are noninvasive, 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 pun1 Delta mutants misregulate amino acid biosynthetic genes during nitrogen stress. By chromatin immunoprecipitation and reverse transcription-PCR, we find that the filamentous growth factor Mss11p directly binds the PUN1 promoter and regulates its transcription. In total, this study provides the first profile of differential protein abundance during pseudohyphal growth, identifying a previously uncharacterized membrane compartment of Can1 protein required for wild-type nitrogen signaling and filamentous growth.