title: A Comprehensive Membrane Interactome Mapping of Sho1p Reveals Fps1p as a Novel Key Player in the Regulation of the HOG Pathway in S. cerevisiae.
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.
authors: Donnelly Centre, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada.
authors: Mandy Hiu Yi
authors: MH
authors: Lam
authors: Donnelly Centre, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada.
authors: Jamie
authors: J
authors: Snider
authors: 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.
authors: Monique
authors: M
authors: Rehal
authors: Donnelly Centre, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada.
authors: Victoria
authors: V
authors: Wong
authors: Donnelly Centre, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada.
authors: Farzaneh
authors: F
authors: Aboualizadeh
authors: Donnelly Centre, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada.
authors: Luka
authors: L
authors: Drecun
authors: Donnelly Centre, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada.
authors: Olivia
authors: O
authors: Wong
authors: Donnelly Centre, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada.
authors: Bellal
authors: B
authors: Jubran
authors: Donnelly Centre, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada.
authors: Meirui
authors: M
authors: Li
authors: Donnelly Centre, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada.
authors: Mehrab
authors: M
authors: Ali
authors: Department of Biochemistry, Research and Innovation Centre, University of Regina, 3737 Wascana Parkway, Regina, SK S4S 0A2, Canada.
authors: Matthew
authors: M
authors: Jessulat
authors: Department of Biochemistry, Research and Innovation Centre, University of Regina, 3737 Wascana Parkway, Regina, SK S4S 0A2, Canada.
authors: Viktor
authors: V
authors: Deineko
authors: Department of Molecular Genetics, The Ohio State University, 318 West 12th Avenue, Columbus, OH 43210, USA.
authors: Rachel
authors: R
authors: Miller
authors: Molecular Cellular Developmental Biology Program, The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210, USA.
authors: Mid eum
authors: Me
authors: Lee
authors: 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.
authors: Hay-Oak
authors: HO
authors: Park
authors: 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.
authors: Alan
authors: A
authors: Davidson
authors: Department of Biochemistry, Research and Innovation Centre, University of Regina, 3737 Wascana Parkway, Regina, SK S4S 0A2, Canada.
authors: Mohan
authors: M
authors: Babu
authors: 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.
authors: Igor
authors: I
authors: Stagljar
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D005990:Glycerol / Q000378:metabolism; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D009994:Osmolar Concentration; D010766:Phosphorylation; D025941:Protein Interaction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D018909:src Homology Domains
chemical_list: C093263:FPS1 protein, S cerevisiae; D008565:Membrane Proteins; C459787:SHO1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C088589:HOG1 protein, S cerevisiae; D020928:Mitogen-Activated Protein Kinases; D005990:Glycerol
keywords: MAPK pathways; SH3 domains; high osmolarity glycerol (HOG) pathway; membrane yeast two-hybrid assay; protein–protein interactions
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