Effects Of Phosphatases And Kinases To The Subcellular Localization And Function Of Rim101 In Saccharomyces Cerevisiae

Saccharomyces cerevisiae Rim101, is a zinc-finger transcriptional factor that functions in the response of Saccharomyces cerevisiae cells to both alkaline and salt stress, calcium homeostasis, cell differentiation and selenite toxicity. Research the Rim101 regulation system make the molecular mechanism more clearly that Saccharomyces cerevisiae cells adapt to alkaline conditions. The subcellular localization of Rim101 is related to its function, the cutting form and located in the nucleus is the functional protein. deleting the cyclin-dependent kinase gene PHO85 leads to the nuclear accumulation of Rim101. To identify new regulatory factors in the subcellular localization of Rim101, we have screened deletion mutants of non-essential genes encoding 73 phosphatases or 139 kinases through a fluorescent microscopy approach. We demonstrate that the thosphatidylinositol phosphate(Ptd InsP) phosphatase Sac1 regulates the subcellular localization of Rim101. Further studies show that deletion of SAC1 leads to elevation of cytosolic free calcium level, increase the activation level of calcium/calcineurin signaling, which led to the activation of ENA1(the gene of coding H+-ATP enzyme). Deletion of SAC1 was sensitive to lithium and the induction of pHAC111-Rim101-531, the constitutively active form of Rim101, partially suppressed this lithium sensitivity. Immunoblot analysis showed that 3HA-Rim101 processing level in the sac1 had no significant difference from that of the wild type. In addition, the expression level of Nrg1-HA in the sac1 was not significantly different from that of the wild type. These results indicate that the deletion of SAC1 increase the Rim101 nuclear accumulation but does not affect its processing. Sac1 might negative control the formation of functional Rim101.To wonder whether the deletion of phosphatase or kinase genes affect the function of Rim101, we tested lithium sensitivity of all the mutants. Our results showed that mutations of 9 genes encoding in phosphatase and 13 genes involved in kinase were sensitive to 0.4 M LiCl. Among these mutants, the sensitivity of 0.4 mol?L-1 LiCl of 6 gene mutants encoding for phosphatase, bud14, rts1, ptp3, inp51, siw14, oca1, and 6 genes mutants encoding for kinase, cka2, yck1, sat4, lcb3, hal5, ipk1 can be restored by introducing the pHAC111-RIM101-531 plasmid containing a RIM101 gene version truncated at the codon 531. Further studies showed that the expression of ENA1-Lac Z is reduced only in 6 mutations of siw14, rts1, oca1, ipk1, lcb3 and hal5 compared to the wild type, while the activities of ENA1-Lac Z in other mutations are similar to, or even higher than that of the wild type. These results indicate that those phosphatases or kinases regulate the Rim101 pathway and the lithium tolerance by different mechanisms.