Hph1p and Hph2p, Novel Components of Calcineurin-Mediated Stress Responses in Saccharomyces cerevisiae

Abstract

Calcineurin is a Ca2؉ -and calmodulin-dependent protein phosphatase that plays a key role in animal and yeast physiology. In the yeast Saccharomyces cerevisiae, calcineurin is required for survival during several environmental stresses, including high concentrations of Na ؉ , Li ؉ , and Mn 2؉ ions and alkaline pH. One role of calcineurin under these conditions is to activate gene expression through its regulation of the Crz1p transcription factor. We have identified Hph1p as a novel substrate of calcineurin. HPH1 (YOR324C) and its homolog HPH2 (YAL028W) encode tail-anchored integral membrane proteins that interact with each other in the yeast two-hybrid assay and colocalize to the endoplasmic reticulum. Hph1p and Hph2p serve redundant roles in promoting growth under conditions of high salinity, alkaline pH, and cell wall stress. Calcineurin modifies the distribution of Hph1p within the endoplasmic reticulum and is required for full Hph1p activity in vivo. Furthermore, calcineurin directly dephosphorylates Hph1p and interacts with it through a sequence motif in Hph1p, PVIAVN. This motif is related to calcineurin docking sites in other substrates, such as NFAT and Crz1p, and is required for regulation of Hph1p by calcineurin. In contrast, Hph2p neither interacts with nor is dephosphorylated by calcineurin. Ca 2؉ -induced Crz1p-mediated transcription is unaffected in hph1⌬ hph2⌬ mutants, and genetic analyses indicate that HPH1/HPH2 and CRZ1 act in distinct pathways downstream of calcineurin. Thus, Hph1p and Hph2p are components of a novel Ca 2؉ -and calcineurin-regulated response required to promote growth under conditions of high Na ؉ , alkaline pH, and cell wall stress.

Targeting of Calcineurin to an NFAT-like Docking Site Is Required for the Calcium-dependent Activation of the Background K+ Channel, TRESK

“…Several proteins are substrates of calcineurin (24); however, only a small subset of them interacts directly with the calcium/calmodulin-dependent phosphatase through an NFAT-like docking site (13,14,(25)(26)(27). Previously, we demonstrated unequivocally that the calciumdependent regulation of the two-pore domain K ϩ channel, TRESK, is mediated by calcineurin (3).…”

A Genome-Wide Enhancer Screen Implicates Sphingolipid Composition in Vacuolar ATPase Function in Saccharomyces cerevisiae

“…HPH1 has a homolog in S. cerevisiae, HPH2, and both Hph1p and Hph2p reside in the ER membrane (Heath et al 2004). It has been reported that deletions in HPH1 and HPH2 display a synthetic growth defect under alkaline conditions of pH 8.8 (Heath et al 2004). Since a reduction in V-ATPase function results in increased sensitivity to calcium or zinc, we determined whether a loss of the HPH genes results in a metal-specific phenotype.…”

“…vma21QQ voa1D yeast showed a compromised level of growth under these conditions and a deletion of HPH1 in this strain caused a further increase in sensitivity (Figure 2A). HPH1 has a homolog in S. cerevisiae, HPH2, and both Hph1p and Hph2p reside in the ER membrane (Heath et al 2004). It has been reported that deletions in HPH1 and HPH2 display a synthetic growth defect under alkaline conditions of pH 8.8 (Heath et al 2004).…”

“…We chose to examine Orm2p for further study because it was an ER-localized, integral membrane protein. Also, Hph1p has been identified as an ER-localized binding partner of calcineurin (Heath et al 2004). Due to the genetic link between calcineurin and the V-ATPase (Tanida et al 1995), we also chose HPH1 for further study.…”

“…We chose to characterize two gene families, HPH1 and ORM2, whose protein products had been previously reported to localize to the ER; both have homologs within budding yeast, HPH2 and ORM1, respectively (Hjelmqvist et al 2002;Heath et al 2004). HPH1 and HPH2 have been characterized as new components of calcineurin signaling (Heath et al 2004).…”