title: Binding of the Extracellular Eight-Cysteine Motif of Opy2 to the Putative Osmosensor Msb2 Is Essential for Activation of the Yeast High-Osmolarity Glycerol Pathway.
abstract: To adapt to environmental high osmolarity, the budding yeast Saccharomyces cerevisiae activates the Hog1 mitogen-activated protein kinase, which regulates diverse osmoadaptive responses. Hog1 is activated through the high-osmolarity glycerol (HOG) pathway, which consists of independent upstream signaling routes termed the SLN1 branch and the SHO1 branch. Here, we report that the extracellular cysteine-rich (CR) domain of the transmembrane-anchor protein Opy2 binds to the Hkr1-Msb2 homology (HMH) domain of the putative osmosensor Msb2 and that formation of the Opy2-Msb2 complex is essential for osmotic activation of Hog1 through the MSB2 subbranch of the SHO1 branch. By analyzing the phenotypes of mutants with Opy2 cysteine-to-alanine mutations, we deduced that the CR domain forms four intramolecular disulfide bonds. To probe for the potential induction of conformational changes in the Opy2-Msb2 complex by osmostress, we constructed mutants with a site-specific Cys-to-Ala mutation of the Opy2 CR domain and mutants with a Cys substitution of the Msb2 HMH domain. Each of these mutants had a reduced cysteine. These mutants were then combinatorially cross-linked using chemical cross-linkers of different lengths. Cross-linking between Opy2 Cys48 and Msb2 Cys1023 was sensitive to osmotic changes, suggesting that osmostress induced a conformational change. We therefore propose that the Opy2-Msb2 complex might serve as an osmosensor.
authors.affiliation: Division of Molecular Cell Signaling, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
authors.forename: Katsuyoshi
authors.initials: K
authors.lastname: Yamamoto
authors.affiliation: Division of Molecular Cell Signaling, Institute of Medical Science, University of Tokyo, Tokyo, Japan tategone@ims.u-tokyo.ac.jp h-saito@ims.u-tokyo.ac.jp.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Tatebayashi
authors.affiliation: Division of Molecular Cell Signaling, Institute of Medical Science, University of Tokyo, Tokyo, Japan tategone@ims.u-tokyo.ac.jp h-saito@ims.u-tokyo.ac.jp.
authors.forename: Haruo
authors.initials: H
authors.lastname: Saito
mesh_terms: D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D009997:Osmotic Pressure; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D060066:Protein Interaction Maps; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D015398:Signal Transduction
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; C076360:MSB2 protein, S cerevisiae; D008565:Membrane Proteins; C547524:Opy2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C088589:HOG1 protein, S cerevisiae; D020928:Mitogen-Activated Protein Kinases; D003545:Cysteine; D005990:Glycerol