Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
J Biosci Bioeng. 2011 Mar;111(3):249-54. doi: 10.1016/j.jbiosc.2010.11.013. Epub 2011 Jan 14.
A Saccharomyces cerevisiae mutant lacking PPZ1, encoding a serine/threonine protein phosphatase (PPase), is caffeine-sensitive. To clarify the function of Ppz1 in resistance to caffeine, we attempted systematically to identify protein kinase (PKase) whose disruption lead to suppression of caffeine sensitive phenotype of the ∆ppz1 disruptant since disruption of PPZ1 might cause caffeine sensitivity by increasing its phosphorylated substrates and we presumed that disruption of genes for PKase sharing the substrate with Ppz1 could restore the resistance through bypassing necessity for dephosphorylation of substrates. Among the 102 viable pkase disruptions, disruption of either SAT4 or HAL5 suppressed the caffeine sensitivity phenotype and increased expression of ENA1, encoding a P-type ATPase of the ∆ppz1 disruptant. Because increased expression of ENA1 in the ∆ppz1 disruptant was found to be suppressed by disruption of GLN3, localization and phosphorylation of Gln3 in the ∆ppz1 disruptant was compared to that in the ∆ppz1∆sat4 and ∆ppz1∆hal5 double disruptants. Gln3 was found to accumulate in the nucleus in the ∆ppz1 disruptant, and this nuclear localization was abolished by disruption of either SAT4 or HAL5. Interestingly, the level of Gln3 phosphorylation in the ∆ppz1∆sat4 and ∆ppz1∆hal5 disruptants decreased relative to wild type independent of caffeine. From these observations, we conclude that Ppz1 controls Gln3 localization by regulating its phosphorylation state in combination with Sat4 and Hal5.
酿酒酵母 ppz1 缺失突变体（编码丝氨酸/苏氨酸蛋白磷酸酶）对咖啡因敏感。为了阐明 Ppz1 在咖啡因抗性中的功能，我们试图系统地鉴定蛋白激酶（PKase），其缺失导致 ∆ppz1 缺失突变体的咖啡因敏感表型得到抑制，因为 PPZ1 的缺失可能通过增加其磷酸化底物而导致咖啡因敏感性，并且我们推测与 Ppz1 具有共同底物的 PKase 基因的缺失可以通过绕过底物去磷酸化的必要性来恢复抗性。在 102 个可行的 pkase 缺失中，sat4 或 hal5 的缺失抑制了 ∆ppz1 缺失突变体的咖啡因敏感性表型，并增加了 ENA1 的表达，该基因编码 P 型 ATPase。由于在 ∆ppz1 缺失突变体中发现 ENA1 的表达增加被 GLN3 的缺失所抑制，因此比较了 ∆ppz1 缺失突变体中 Gln3 的定位和磷酸化与 ∆ppz1∆sat4 和 ∆ppz1∆hal5 双缺失突变体中的定位和磷酸化。发现 Gln3 在 ∆ppz1 缺失突变体中积累在核内，并且这种核定位被 sat4 或 hal5 的缺失所消除。有趣的是，Gln3 磷酸化水平在 ∆ppz1∆sat4 和 ∆ppz1∆hal5 缺失突变体中相对于野生型独立于咖啡因而降低。从这些观察结果中，我们得出结论，Ppz1 通过与 Sat4 和 Hal5 一起调节其磷酸化状态来控制 Gln3 的定位。
