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.

[1]  E. de Nadal,et al.  Osmostress‐induced gene expression – a model to understand how stress‐activated protein kinases (SAPKs) regulate transcription , 2015, The FEBS journal.

[2]  G. Ammerer,et al.  Msb2 is a Ste11 membrane concentrator required for full activation of the HOG pathway. , 2015, Biochimica et biophysica acta.

[3]  H. Saito,et al.  Osmosensing and scaffolding functions of the oligomeric four-transmembrane domain osmosensor Sho1 , 2015, Nature Communications.

[4]  Hema Adhikari,et al.  Role of the Unfolded Protein Response in Regulating the Mucin-Dependent Filamentous-Growth Mitogen-Activated Protein Kinase Pathway , 2015, Molecular and Cellular Biology.

[5]  H. Saito,et al.  Yeast Osmosensors Hkr1 and Msb2 Activate the Hog1 MAPK Cascade by Different Mechanisms , 2014, Science Signaling.

[6]  P. Cullen,et al.  The Filamentous Growth MAPK Pathway Responds to Glucose Starvation Through the Mig1/2 Transcriptional Repressors in Saccharomyces cerevisiae , 2012, Genetics.

[7]  Francesc Posas,et al.  Response to Hyperosmotic Stress , 2012, Genetics.

[8]  Kevin W Eliceiri,et al.  NIH Image to ImageJ: 25 years of image analysis , 2012, Nature Methods.

[9]  T. Kupka,et al.  M-Track: detecting short-lived protein-protein interactions in vivo , 2012, Nature Methods.

[10]  V. Smith.  Phylogeny of whey acidic protein (WAP) four-disulfide core proteins and their role in lower vertebrates and invertebrates. , 2011, Biochemical Society transactions.

[11]  C. Bingle.  Towards defining the complement of mammalian WFDC-domain-containing proteins. , 2011, Biochemical Society transactions.

[12]  H. Saito,et al.  Dynamic control of yeast MAP kinase network by induced association and dissociation between the Ste50 scaffold and the Opy2 membrane anchor. , 2010, Molecular cell.

[13]  T. Sulea,et al.  Binding the atypical RA domain of Ste50p to the unfolded Opy2p cytoplasmic tail is essential for the high-osmolarity glycerol pathway. , 2009, Molecular biology of the cell.

[14]  T. Petersen,et al.  A generic method for assignment of reliability scores applied to solvent accessibility predictions , 2009, BMC Structural Biology.

[15]  P. Cullen,et al.  The signaling mucins Msb2 and Hkr1 differentially regulate the filamentation mitogen-activated protein kinase pathway and contribute to a multimodal response. , 2009, Molecular biology of the cell.

[16]  Kazuo Tatebayashi,et al.  Glycosylation defects activate filamentous growth Kss1 MAPK and inhibit osmoregulatory Hog1 MAPK , 2009, The EMBO journal.

[17]  D. Krysan,et al.  Cleavage of the signaling mucin Msb2 by the aspartyl protease Yps1 is required for MAPK activation in yeast , 2008, The Journal of cell biology.

[18]  Kazuo Tatebayashi,et al.  Phosphorylated Ssk1 Prevents Unphosphorylated Ssk1 from Activating the Ssk2 Mitogen-Activated Protein Kinase Kinase Kinase in the Yeast High-Osmolarity Glycerol Osmoregulatory Pathway , 2008, Molecular and Cellular Biology.

[19]  H. Saito,et al.  Two Adjacent Docking Sites in the Yeast Hog1 Mitogen-Activated Protein (MAP) Kinase Differentially Interact with the Pbs2 MAP Kinase Kinase and the Ptp2 Protein Tyrosine Phosphatase , 2008, Molecular and Cellular Biology.

[20]  Kazuo Tatebayashi,et al.  Transmembrane mucins Hkr1 and Msb2 are putative osmosensors in the SHO1 branch of yeast HOG pathway , 2007, The EMBO journal.

[21]  Kazuo Tatebayashi,et al.  Adaptor functions of Cdc42, Ste50, and Sho1 in the yeast osmoregulatory HOG MAPK pathway , 2006, The EMBO journal.

[22]  N. Grishin,et al.  Structural classification of small, disulfide-rich protein domains. , 2006, Journal of molecular biology.

[23]  Rachel E. Lamson,et al.  Dual Role for Membrane Localization in Yeast MAP Kinase Cascade Activation and Its Contribution to Signaling Fidelity , 2006, Current Biology.

[24]  David Y. Thomas,et al.  Adaptor protein Ste50p links the Ste11p MEKK to the HOG pathway through plasma membrane association. , 2006, Genes & development.

[25]  J. Thorner,et al.  The RA Domain of Ste50 Adaptor Protein Is Required for Delivery of Ste11 to the Plasma Membrane in the Filamentous Growth Signaling Pathway of the Yeast Saccharomyces cerevisiae , 2006, Molecular and Cellular Biology.

[26]  Lina L. Feng,et al.  Evolution of distinct EGF domains with specific functions , 2005, Protein science : a publication of the Protein Society.

[27]  Lee Bardwell,et al.  A signaling mucin at the head of the Cdc42- and MAPK-dependent filamentous growth pathway in yeast. , 2004, Genes & development.

[28]  Wendell A. Lim,et al.  Optimization of specificity in a cellular protein interaction network by negative selection , 2003, Nature.

[29]  J. Gorman,et al.  Protein Disulfide Bond Determination by Mass Spectrometry , 2003 .

[30]  Kazuo Tatebayashi,et al.  A docking site determining specificity of Pbs2 MAPKK for Ssk2/Ssk22 MAPKKKs in the yeast HOG pathway , 2003, The EMBO journal.

[31]  M. Ramezani-Rad.  The role of adaptor protein Ste50-dependent regulation of the MAPKKK Ste11 in multiple signalling pathways of yeast , 2003, Current Genetics.

[32]  Sean M. O'Rourke,et al.  A Third Osmosensing Branch in Saccharomyces cerevisiae Requires the Msb2 Protein and Functions in Parallel with the Sho1 Branch , 2002, Molecular and Cellular Biology.

[33]  S. Hohmann.  Osmotic Stress Signaling and Osmoadaptation in Yeasts , 2002, Microbiology and Molecular Biology Reviews.

[34]  J. Gorman,et al.  Protein disulfide bond determination by mass spectrometry. , 2002, Mass spectrometry reviews.

[35]  D. Clarke,et al.  Determining the Dimensions of the Drug-binding Domain of Human P-glycoprotein Using Thiol Cross-linking Compounds as Molecular Rulers* , 2001, The Journal of Biological Chemistry.

[36]  J. Nathans,et al.  Insights into Wnt binding and signalling from the structures of two Frizzled cysteine-rich domains , 2001, Nature.

[37]  M. Whiteway,et al.  Functional characterization of the interaction of Ste50p with Ste11p MAPKKK in Saccharomyces cerevisiae. , 1999, Molecular biology of the cell.

[38]  H. Saito,et al.  A Family of Stress-Inducible GADD45-like Proteins Mediate Activation of the Stress-Responsive MTK1/MEKK4 MAPKKK , 1998, Cell.

[39]  Francesc Posas,et al.  Requirement of STE50 for Osmostress-Induced Activation of the STE11 Mitogen-Activated Protein Kinase Kinase Kinase in the High-Osmolarity Glycerol Response Pathway , 1998, Molecular and Cellular Biology.

[40]  F. Posas,et al.  Osmotic activation of the HOG MAPK pathway via Ste11p MAPKKK: scaffold role of Pbs2p MAPKK. , 1997, Science.

[41]  Francesc Posas,et al.  Yeast HOG1 MAP Kinase Cascade Is Regulated by a Multistep Phosphorelay Mechanism in the SLN1–YPD1–SSK1 “Two-Component” Osmosensor , 1996, Cell.

[42]  T. Maeda,et al.  Activation of yeast PBS2 MAPKK by MAPKKKs or by binding of an SH3-containing osmosensor. , 1995, Science.

[43]  Tatsuya Maeda,et al.  A two-component system that regulates an osmosensing MAP kinase cascade in yeast , 1994, Nature.

[44]  R. Müller,et al.  Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. , 1994, Nucleic acids research.

[45]  E. Winter,et al.  An osmosensing signal transduction pathway in yeast. , 1993, Science.

[46]  G. Boguslawski.  PBS2, a yeast gene encoding a putative protein kinase, interacts with the RAS2 pathway and affects osmotic sensitivity of Saccharomyces cerevisiae. , 1992, Journal of general microbiology.

[47]  J. Broach,et al.  Cloning genes by complementation in yeast. , 1991, Methods in enzymology.

[48]  J. Pringle,et al.  Multicopy suppression of the cdc24 budding defect in yeast by CDC42 and three newly identified genes including the ras-related gene RSR1. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[49]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .