THE YEAST HALOTOLERANCE DETERMINANT HAL3P IS AN INHIBITORY SUBUNIT OFTHE PPZ1P SER THR PROTEIN PHOSPHATASE/

Components of cellular stress responses can be identified by correlati ng changes in stress tolerance with gain or loss of function of define d genes. Previous work has shown that yeast cells deficient in Ppz1 pr otein phosphatase or overexpressing Hal3p, a novel regulatory protein of unknown function, exhibit increased resistance to sodium and lithiu m, whereas cells lacking Hal3p display increased sensitivity. These ef fects are largely a result of changes in expression of ENA1, encoding the major cation extrusion pump of yeast cells. Disruption or overexpr ession 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 P pz1p fused to glutathione S-transferase, and it also copurifies with a ffinity-purified glutathione S-transferase-Ppz1p from yeast extracts. In both cases, the interaction is stronger when only the carboxyl-term inal catalytic phosphatase domain of Ppz1p is expressed. In vitro expe riments reveal that the protein phosphatase activity of Ppz1p is inhib ited by Hal3p. Overexpression of Hal3p suppresses the reduced growth r ate because of the overexpression of Ppz1p and aggravates the lytic ph enotype of a slt2/mpk1 mitogen-activated protein kinase mutant (thus m imicking the deletion of PPZ1), Therefore, Hal3p might modulate divers e physiological functions of the Ppz1 phosphatase, such as salt stress tolerance and cell cycle progression, by acting as a inhibitory subun it.