The unique characteristics of HOG pathway MAPKs in the extremely halotolerant Hortaea werneckii

Abstract

HwHog1A/B, Hortaea werneckii homologues of the MAP kinase Hog1 from Saccharomyces cerevisiae, are vital for the extreme halotolerance of H. werneckii. In mesophilic S. cerevisiae, Hog1 is phosphorylated already at low osmolyte concentrations, and regulates expression of a similar set of genes independent of osmolyte type.To understand how HwHog1 kinases activity is regulated in H. werneckii, we studiedHwHog1A/B activation in vivo, by following phosphorylation of HwHog1A/B in H. werneckii exposed to various osmolytes, and in vitro, by measuring kinase activitiesofrecombinant HwHog1A, HwHog1B and Hog1ΔC. To this end, highly pure and solublerecombinant Hog1 homologues were isolated from insect cells. Our results demonstratethat HwHog1A/B are, in general, transiently phosphorylated in cells shocked with ≥3 M osmolyte, yet constitutive phosphorylation is observed at extreme NaCl andKClconcentrations. Importantly, phosphorylation profiles differ depending on theosmolyte type. Additionally, phosphorylated recombinant HwHog1A/B show lower specific kinase activities compared to Hog1ΔC. In summary, HOG pathway MAPKs in the extremelyhalotolerant H. werneckii show unique characteristics compared to S. cerevisiae homologues. The reported findings contribute to defining the key determinants of H.werneckii osmotolerance, which is important for its potential transfer toeconomically relevant microorganisms and crops.