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J Mol Biol. 2015 Jun 5;427(11):2088-103. doi: 10.1016/j.jmb.2015.01.016. Epub 2015 Jan 30.

A Comprehensive Membrane Interactome Mapping of Sho1p Reveals Fps1p as a Novel Key Player in the Regulation of the HOG Pathway in S. cerevisiae.

Author information

1
Donnelly Centre, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada.
2
Donnelly Centre, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada; Department of Biochemistry, University of Toronto, Medical Science Building, 1 King's College Circle, Toronto, ON M5S 1A8, Canada.
3
Department of Biochemistry, Research and Innovation Centre, University of Regina, 3737 Wascana Parkway, Regina, SK S4S 0A2, Canada.
4
Department of Molecular Genetics, The Ohio State University, 318 West 12th Avenue, Columbus, OH 43210, USA.
5
Molecular Cellular Developmental Biology Program, The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210, USA.
6
Department of Molecular Genetics, The Ohio State University, 318 West 12th Avenue, Columbus, OH 43210, USA; Molecular Cellular Developmental Biology Program, The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210, USA.
7
Department of Biochemistry, University of Toronto, Medical Science Building, 1 King's College Circle, Toronto, ON M5S 1A8, Canada; Department of Molecular Genetics, University of Toronto, Medical Sciences Building, 1 King's College Circle, Toronto, ON M5S 1A8, Canada.
8
Donnelly Centre, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada; Department of Biochemistry, University of Toronto, Medical Science Building, 1 King's College Circle, Toronto, ON M5S 1A8, Canada; Department of Molecular Genetics, University of Toronto, Medical Sciences Building, 1 King's College Circle, Toronto, ON M5S 1A8, Canada. Electronic address: igor.stagljar@utoronto.ca.

Abstract

Sho1p, an integral membrane protein, plays a vital role in the high-osmolarity glycerol (HOG) mitogen-activated protein kinase pathway in the yeast Saccharomyces cerevisiae. Activated under conditions of high osmotic stress, it interacts with other HOG pathway proteins to mediate cell signaling events, ensuring that yeast cells can adapt and remain viable. In an attempt to further understand how the function of Sho1p is regulated through its protein-protein interactions (PPIs), we identified 49 unique Sho1p PPIs through the use of membrane yeast two-hybrid (MYTH), an assay specifically suited to identify PPIs of full-length integral membrane proteins in their native membrane environment. Secondary validation by literature search, or two complementary PPI assays, confirmed 80% of these interactions, resulting in a high-quality Sho1p interactome. This set of putative PPIs included both previously characterized interactors, along with a large subset of interactors that have not been previously identified as binding to Sho1p. The SH3 domain of Sho1p was found to be important for binding to many of these interactors. One particular novel interactor of interest is the glycerol transporter Fps1p, which was shown to require the SH3 domain of Sho1p for binding via its N-terminal soluble regulatory domain. Furthermore, we found that Fps1p is involved in the positive regulation of Sho1p function and plays a role in the phosphorylation of the downstream kinase Hog1p. This study represents the largest membrane interactome analysis of Sho1p to date and complements past studies on the HOG pathway by increasing our understanding of Sho1p regulation.

KEYWORDS:

MAPK pathways; SH3 domains; high osmolarity glycerol (HOG) pathway; membrane yeast two-hybrid assay; protein–protein interactions

PMID:
25644660
PMCID:
PMC5331858
DOI:
10.1016/j.jmb.2015.01.016
[Indexed for MEDLINE]
Free PMC Article
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