Rodríguez-González Miriam, Kawasaki Laura, Velázquez-Zavala Nancy, Domínguez-Martín Eunice, Trejo-Medecigo Abraham, Martagón Natalia, Espinoza-Simón Emilio, Vázquez-Ibarra Araceli, Ongay-Larios Laura, Georgellis Dimitris, de Nadal Eulàlia, Posas Francesc, Coria Roberto
Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México, D.F., México.
Departamento de Bioquímica, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México, D.F., México.
Mol Microbiol. 2017 Jun;104(5):822-836. doi: 10.1111/mmi.13664. Epub 2017 Mar 28.
The Kluyveromyces lactis SLN1 phosphorelay system includes the osmosensor histidine kinase Sln1, the phosphotransfer protein Ypd1 and the response regulator Ssk1. Here we show that K. lactis has a functional phosphorelay system. In vitro assays, using a heterologous histidine kinase, show that the phosphate group is accepted by KlYpd1 and transferred to KlSsk1. Upon hyperosmotic stress the phosphorelay is inactivated, KlYpd1 is dephosphorylated in a KlSln1 dependent manner, and only the version of KlSsk1 that lacks the phosphate group interacts with the MAPKKK KlSsk2. Interestingly, inactivation of the KlPtp2 phosphatase in a ΔKlsln1 mutant did not lead to KlHog1 constitutive phosphorylation. KlHog1 can replace ScHog1p and activate the hyperosmotic response in Saccharomyces cerevisiae, and when ScSln1 is inactivated, KlHog1 becomes phosphorylated and induces cell lethality. All these observations indicate that the phosphorelay negatively regulates KlHog1. Nevertheless, in the absence of KlSln1 or KlYpd1, no constitutive phosphorylation is detected and cells are viable, suggesting that a strong negative feedback that is independent of KlPtp2 operates in K. lactis. Compared with S. cerevisiae, K. lactis has only a moderate accumulation of glycerol and fails to produce trehalose under hyperosmotic stress, indicating that regulation of osmolyte production is different in K. lactis.
乳酸克鲁维酵母的SLN1磷酸化信号转导系统包括渗透感受器组氨酸激酶Sln1、磷酸转移蛋白Ypd1和应答调节因子Ssk1。在此我们表明乳酸克鲁维酵母具有功能性磷酸化信号转导系统。使用异源组氨酸激酶进行的体外试验表明，磷酸基团被KlYpd1接受并转移至KlSsk1。在高渗胁迫下，磷酸化信号转导被失活，KlYpd1以依赖KlSln1的方式去磷酸化，并且只有缺乏磷酸基团的KlSsk1版本与MAPKKK KlSsk2相互作用。有趣的是，在ΔKlsln1突变体中KlPtp2磷酸酶的失活并未导致KlHog1组成型磷酸化。KlHog1可以替代ScHog1p并激活酿酒酵母中的高渗应答，并且当ScSln1失活时，KlHog1会被磷酸化并诱导细胞致死。所有这些观察结果表明磷酸化信号转导对KlHog1起负调控作用。然而，在缺乏KlSln1或KlYpd1的情况下，未检测到组成型磷酸化且细胞仍可存活，这表明在乳酸克鲁维酵母中存在一种独立于KlPtp2的强大负反馈机制。与酿酒酵母相比，乳酸克鲁维酵母仅适度积累甘油，并且在高渗胁迫下无法产生海藻糖，这表明乳酸克鲁维酵母中渗透溶质产生的调控有所不同。
