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A PhotoClick cholesterol‐based quantitative proteomics screen for cytoplasmic sterol‐binding proteins in Saccharomyces cerevisiae
Author(s) -
Chauhan Neha,
Sere Yves Y.,
Sokol Anna M.,
Graumann Johannes,
Me Anant K.
Publication year - 2020
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.3448
Subject(s) - biology , sterol , saccharomyces cerevisiae , ergosterol , biochemistry , cytosol , membrane protein , proteomics , microbiology and biotechnology , endoplasmic reticulum , proteome , transport protein , yeast , cholesterol , gene , membrane , enzyme
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 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 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 chemoproteomic strategy to identify all cytosolic SBPs. We generated a cytosol fraction enriched in SBPs and captured the proteins with a photoreactive clickable cholesterol analogue. 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 orthologues 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.