Kejžar Anja, Cibic Matej, Grøtli Morten, Plemenitaš Ana, Lenassi Metka
Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov Trg 2, SI-1000 Ljubljana, Slovenia.
Department of Chemistry and Molecular Biology, University of Gothenburg, SE-412 96 Gothenburg, Sweden.
FEMS Microbiol Lett. 2015 Apr;362(8):fnv046. doi: 10.1093/femsle/fnv046. Epub 2015 Mar 30.
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 studied HwHog1A/B activation in vivo, by following phosphorylation of HwHog1A/B in H. werneckii exposed to various osmolytes, and in vitro, by measuring kinase activities of recombinant HwHog1A, HwHog1B and Hog1ΔC. To this end, highly pure and soluble recombinant Hog1 homologues were isolated from insect cells. Our results demonstrate that HwHog1A/B are, in general, transiently phosphorylated in cells shocked with ≥3 M osmolyte, yet constitutive phosphorylation is observed at extreme NaCl and KCl concentrations. Importantly, phosphorylation profiles differ depending on the osmolyte type. Additionally, phosphorylated recombinant HwHog1A/B show lower specific kinase activities compared to Hog1ΔC. In summary, HOG pathway MAPKs in the extremely halotolerant 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 to economically relevant microorganisms and crops.
HwHog1A/B是来自酿酒酵母的丝裂原活化蛋白激酶Hog1在威尼克嗜盐霉菌（Hortaea werneckii）中的同源物，对威尼克嗜盐霉菌的极端耐盐性至关重要。在嗜温的酿酒酵母中，Hog1在低渗透压浓度下就会被磷酸化，并独立于渗透压类型调节一组相似基因的表达。为了了解HwHog1激酶活性在威尼克嗜盐霉菌中是如何被调控的，我们通过检测暴露于各种渗透压下的威尼克嗜盐霉菌中HwHog1A/B的磷酸化情况，在体内研究了HwHog1A/B的激活，并且通过测量重组HwHog1A、HwHog1B和Hog1ΔC的激酶活性，在体外进行了研究。为此，从昆虫细胞中分离出了高度纯净且可溶的重组Hog1同源物。我们的结果表明，一般来说，HwHog1A/B在受到≥3M渗透压冲击的细胞中会被短暂磷酸化，但在极端的NaCl和KCl浓度下会观察到组成型磷酸化。重要的是，磷酸化谱因渗透压类型而异。此外，与Hog1ΔC相比，磷酸化的重组HwHog1A/B显示出较低的比激酶活性。总之，与酿酒酵母同源物相比，极端耐盐的威尼克嗜盐霉菌中的HOG途径丝裂原活化蛋白激酶表现出独特的特征。所报道的研究结果有助于确定威尼克嗜盐霉菌耐渗透压的关键决定因素，这对于将其潜在转移到经济相关的微生物和作物中很重要。
