A Comprehensive Membrane Interactome Mapping of Sho1p Reveals Fps1p as a Novel Key Player in the Regulation of the HOG Pathway in S. cerevisiae Mandy Hiu Yi Lam 1 , Jamie Snider 1 , Monique Rehal 1,2 , Victoria Wong 1 , Farzaneh Aboualizadeh 1 , Luka Drecun 1 , Olivia Wong 1 , Bellal Jubran 1 , Meirui Li 1 , Mehrab Ali 1 , Matthew Jessulat 3 , Viktor Deineko 3 , Rachel Miller 4 , Mid eum Lee 5 , Hay-Oak Park 4,5 , Alan Davidson 2,6 , Mohan Babu 3 and Igor Stagljar 1,2,6 1 - Donnelly Centre, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada 2 - 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, University of Toronto, Medical Sciences Building, 1 King's College Circle, Toronto, ON M5S 1A8, Canada Correspondence to Igor Stagljar: Donnelly Centre, University of Toronto, Room 1204, 160 College Street, Toronto, ON M5S 3E1, Canada. igor.stagljar@utoronto.ca http://dx.doi.org/10.1016/j.jmb.2015.01.016 Edited by S. Koide 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. © 2015 Elsevier Ltd. All rights reserved. Introduction Organisms often face various internal and external stresses and, over time, develop systems to adapt to these challenges. The budding yeast Saccharomyces cerevisiae has long been an excellent model to study many different processes, including those associated with stress, in eukaryotic cells. The high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) stress response pathway ensures that yeast cells adapt and remain viable under conditions of high osmotic stress (reviewed in Ref. [1]). Turgor pressure, and therefore cell viability, is lost during conditions under which the concentration of the solute is higher in IMF YJMBI-64677; No. of pages: 16; 4C: 2, 7, 8, 12 0022-2836/© 2015 Elsevier Ltd. All rights reserved. J Mol Biol (2015) xx, xxx–xxx Article Please cite this article as: Lam Mandy Hiu Yi, et al, A Comprehensive Membrane Interactome Mapping of Sho1p Reveals Fps1p as a Novel Key Player in the Regulation of the HOG Pathway in ..., J Mol Biol (2015), http://dx.doi.org/10.1016/j.jmb.2015.01.016