The yeast halotolerance determinant Hal3p is an inhibitory subunit of the Ppz1p Ser/Thr protein phosphatase
The yeast halotolerance determinant Hal3p is an inhibitory subunit of the Ppz1p Ser/Thr protein phosphatase
复制标题
DOI：
10.1073/pnas.95.13.7357
复制
发表时间：
1998-06-23
影响因子：
11.1
通讯作者：
Ariño, J
中科院分区：
文献类型：
--
作者：
de Nadal, E;Clotet, J;Ariño, J
细胞应激反应的组分可以通过将应激耐受性的变化与确定基因的功能获得或丧失相关联来鉴定。先前的工作表明，缺乏Ppz 1蛋白磷酸酶或过表达Hal 3 p（一种功能未知的新型调节蛋白）的酵母细胞表现出对钠和锂的抗性增加，而缺乏Hal 3 p的细胞则表现出敏感性增加。这些效应主要是ENA 1表达变化的结果，ENA 1编码酵母细胞的主要阳离子挤出泵。HAL 3（也称为SIS 2）的破坏或过表达在PPZ 1不存在的情况下对耐盐性没有影响，这表明Hal 3 p可能在一种新的信号转导途径中在Ppz 1 p的上游起作用。Hal 3 p通过使用与谷胱甘肽S-转移酶融合的固定化、细菌表达的Ppz 1 p从粗酵母匀浆中回收，并且还与来自酵母提取物的亲和纯化的谷胱甘肽S-转移酶-Ppz 1 p共纯化。在这两种情况下，当只有羧基末端的催化磷酸酶结构域的Ppz 1 p表达的相互作用更强。体外实验表明，Hal 3 p抑制Ppz 1 p的蛋白磷酸酶活性。Hal 3 p的过表达抑制了由于Ppz 1 p的过表达而导致的生长速率降低，并使slt 2/mpk 1有丝分裂原活化蛋白激酶突变体的裂解表型（从而模仿PPZ 1的缺失）发生突变。因此，Hal 3 p可能通过作为抑制亚基来调节Ppz 1磷酸酶的多种生理功能，例如作为盐胁迫耐受和细胞周期进程。
Components of cellular stress responses can be identified by correlating changes in stress tolerance with gain or loss of function of defined genes. Previous work has shown that yeast cells deficient in Ppz1 protein phosphatase or overexpressing Hal3p, a novel regulatory protein of unknown function, exhibit increased resistance to sodium and lithium, whereas cells lacking Hal3p display increased sensitivity. These effects are largely a result of changes in expression of ENA1, encoding the major cation extrusion pump of yeast cells. Disruption or overexpression of HAL3 (also known as SIS2) has no effect on salt tolerance in the absence of PPZ1, suggesting that Hal3p might function upstream of Ppz1p in a novel signal transduction pathway. Hal3p is recovered from crude yeast homogenates by using immobilized, bacterially expressed Ppz1p fused to glutathione S-transferase, and it also copurifies with affinity-purified glutathione S-transferase-Ppz1p from yeast extracts. In both cases, the interaction is stronger when only the carboxyl-terminal catalytic phosphatase domain of Ppz1p is expressed. In vitro experiments reveal that the protein phosphatase activity of Ppz1p is inhibited by Hal3p. Overexpression of Hal3p suppresses the reduced growth rate because of the overexpression of Ppz1p and aggravates the lytic phenotype of a slt2/mpk1 mitogen-activated protein kinase mutant (thus mimicking the deletion of PPZ1), Therefore, Hal3p might modulate diverse physiological functions of the Ppz1 phosphatase, such as salt stress tolerance and cell cycle progression, by acting as a inhibitory subunit.