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 sub-branch of the SHO1 branch. By analyzing the phenotypes of Opy2 cysteine-to-alanine mutants, we deduced that the CR domain forms four intramolecular disulfide bonds. To probe for potential induction of conformational changes in the Opy2-Msb2 complex by osmostress, we constructed site-specific Cys-to-Ala mutants of the Opy2 CR domain and Cys substitution mutants of the Msb2 HMH domain. Each of these mutants has a reduced cysteine. These mutants were then combinatorially crosslinked using chemical crosslinkers of different lengths. Crosslinking 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.