Dataset Information

A PhotoClick cholesterol-based quantitative proteomics screen for cytoplasmic sterol binding proteins in Saccharomyces cerevisiae

ABSTRACT: Ergosterol is a prominent component of the yeast plasma membrane and essential for yeast cell viability. It is synthesized in the endoplasmic reticulum and transported to the plasma membrane by non-vesicular mechanisms requiring carrier proteins. Oxysterol binding protein homologues and yeast StARkin proteins have been proposed to function as sterol carriers. Although many of these proteins are capable of transporting sterols between synthetic lipid vesicles in vitro, they are not essential for ergosterol transport in cells, indicating that they may be functionally redundant with each other or with additional - as yet unidentified - sterol carriers. To address this point we hypothesized that sterol transport proteins are also sterol binding proteins (SBPs), and used an in vitro chemo-proteomic strategy to identify all cytosolic SBPs. We generated a cytosol fraction enriched in SBPs and captured the proteins with a photoreactive clickable cholesterol analog. Quantitative proteomics of the captured proteins identified 342 putative SBPs. Analysis of these identified proteins based on their annotated function, reported drug phenotypes, interactions with proteins regulating lipid metabolism, gene ontology, and presence of mammalian orthologs revealed a subset of 62 characterized and nine uncharacterized candidates. Five of the uncharacterized proteins play a role in maintaining plasma membrane integrity as their absence affects the ability of cells to grow in the presence of nystatin or myriocin. We anticipate that the dataset reported here will be a comprehensive resource for functional analysis of sterol binding/transport proteins and provide insights into novel aspects of non-vesicular sterol trafficking.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Johannes Graumann

LAB HEAD: Anant K. Menon

PROVIDER: PXD014555 | Pride | 2019-11-25

REPOSITORIES: Pride

ACCESS DATA

Publications

A PhotoClick cholesterol-based quantitative proteomics screen for cytoplasmic sterol-binding proteins in Saccharomyces cerevisiae.

Chauhan Neha N Sere Yves Y YY Sokol Anna M AM Graumann Johannes J Menon Anant K AK

Yeast (Chichester, England) 20200101 1

Ergosterol is a prominent component of the yeast plasma membrane and essential for yeast cell viability. It is synthesized in the endoplasmic reticulum and transported to the plasma membrane by nonvesicular mechanisms requiring carrier proteins. Oxysterol-binding protein homologues and yeast StARkin proteins have been proposed to function as sterol carriers. Although many of these proteins are capable of transporting sterols between synthetic lipid vesicles in vitro, they are not essential for e ...[more]

PMID: 31758572

Similar Datasets

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.

Dataset Information

A PhotoClick cholesterol-based quantitative proteomics screen for cytoplasmic sterol binding proteins in Saccharomyces cerevisiae

Publications

A PhotoClick cholesterol-based quantitative proteomics screen for cytoplasmic sterol-binding proteins in Saccharomyces cerevisiae.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure