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.
ACKNOWLEDGMENTS
We thank Hiroyuki Fukuda and Akiko Nishimura for helpful discussions, Miho Nagoya for her excellent technical assistance, and Pauline O'Grady for her critical reading and editing of the manuscript.
We have no competing financial interests to declare.