The yeast halotolerance determinant Hal3p is an inhibitory subunit of the Ppz1p Ser/Thr protein phosphatase

Abstract

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 slt2͞mpk1 mitogenactivated 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.

Protein phosphatase CaPpz1 is involved in cation homeostasis, cell wall integrity and virulence of Candida albicans

“…slt2 cells are prone to lysis and are very sensitive to high temperature, caffeine, or cell wall-damaging compounds. Overexpression of ScPPZ1 suppresses the lytic phenotype of an slt2 mutant, whereas deletion of the ScPPZ1 gene or inhibition of its phosphatase activity results in a phenotype additive to that of the slt2 strain (de Nadal et al, 1998;Lee et al, 1993). The functional interaction between ScPpz1 and the CWI pathway has been explained on the basis of the combination of increased internal turgor pressure in Ppz-deficient strains and cell wall instability observed in strains lacking Slt2 (Merchan et al, 2004).…”

“…Therefore, in budding yeast, the Ppz proteins play key roles in cation homeostasis, which is likely to affect cell cycle regulation. The Hal3 and Vhs3 regulatory subunits bind to the catalytic domain of ScPpz1 and inhibit its activity (de Nadal et al, 1998;Ruiz et al, 2004). The overexpression of these inhibitors mimics the effects of the scppz1 mutation.…”

Functional Characterization of the Yeast Ppz1 Phosphatase Inhibitory Subunit Hal3

“…In contrast, besides the early observation that the characteristic highly acidic tail of Hal3 is required for function in vivo (7,8), very little is known regarding the structural elements defining the cellular role of Hal3 or its function as phosphatase inhibitor. Interestingly, Hal3 does not structurally resemble previously characterized PP1c phosphatase inhibitors and it does not bind or inhibit in vitro yeast PP1c (encoded by GLC7 in S. cerevisiae) (9,21). Therefore, previous experience and knowledge on the regulation of Glc7 by its diverse regulatory subunits were of little help to face the question of how Hal3 might bind to and inhibit Ppz1.…”

“…sit4 and hal3 mutations display synthetic lethality due to G 1 blockade, whereas high-copy expression of HAL3 accelerates entry into S phase after an ␣-factor-induced G 1 arrest in a sit4 mutant (7,10). Finally, high-copy expression of Hal3 aggravates the lytic phenotype of a Slt2/Mpk1 MAP kinase mutant, whereas, in contrast, a lack of HAL3 improves growth of this strain (9).…”

“…Hal3 acts as a negative regulatory subunit of Ppz1 by binding to the phosphatase carboxyl-terminal catalytic domain (9) and strongly inhibits Ppz1 activity, thus modulating its diverse physiological functions. Therefore, the overexpression of Hal3 provides increased salt tolerance, whereas hal3 cells are hypersensitive to sodium and lithium cations.…”