The Golgi Ca2+-ATPase KlPmr1p function is required for oxidative stress response by controlling the expression of the heat-shock element HSP60 in Kluyveromyces lactis

The Golgi P-type Ca2+-ATPase, Pmr1p, is the major player for calcium homeostasis in yeast. The inactivation of KlPMR1 in Kluyveromyces lactis leads to high pleiotropic phenotypes that include reduced glycosylation, cell wall defects, and alterations of mitochondrial metabolism. In this article we found that cells lacking KlPmr1p have a morphologically altered mitochondrial network and that mitochondria (m) from Klpmr1delta cells accumulate Ca2+ more slowly and reach a lower [Ca2+]m level, when exposed to [Ca2+] < 5 microM, than wild-type cells. The Klpmr1delta cells also exhibit traits of ongoing oxidative stress and present hyperphosphorylation of KlHog1p, the hallmark for the activation of stress response pathways. The mitochondrial chaperone KlHsp60 acts as a multicopy suppressor of phenotypes that occur in cells lacking the Ca2+-ATPase, including relief from oxidative stress and recovery of cell wall thickness and functionality. Inhibition of KlPMR1 function decreases KlHSP60 expression at both mRNA and protein levels. Moreover, KlPRM1 loss of function correlates with both decreases in HSF DNA binding activity and KlHSP60 expression. We suggest a role for KlPMR1 in HSF DNA binding activity, which is required for proper KlHSP60 expression, a key step in oxidative stress response.     

Mitogen-activated protein kinase stimulation of Ca(2+) signaling is required for survival of endoplasmic reticulum stress in yeast

Endoplasmic reticulum (ER) stress in the budding yeast Saccharomyces cerevisiae triggers Ca2+ influx through a plasma membrane channel composed of Cch1 and Mid1. This response activates calcineurin, which helps to prevent cell death during multiple forms of ER stress, including the response to azole-class antifungal drugs. Herein, we show that ER stress activates the cell integrity mitogen-activate protein kinase cascade in yeast and that the activation of Pkc1 and Mpk1 is necessary for stimulation of the Cch1-Mid1 Ca2+ channel independent of many known targets of Mpk1 (Rlm1, Swi4, Swi6, Mih1, Hsl1, and Swe1). ER stress generated in response to miconazole, tunicamycin, or other inhibitors also triggered a transient G2/M arrest that depended upon the Swe1 protein kinase. Calcineurin played little role in the Swe1-dependent cell cycle arrest and Swe1 had little effect on calcineurin-dependent avoidance of cell death. These findings help to clarify the interactions between Mpk1, calcineurin, and Swe1 and suggest that the calcium cell survival pathway promotes drug resistance independent of both the unfolded protein response and the G2/M cell cycle checkpoint.     

Septin-dependent assembly of a cell cycle-regulatory module in Saccharomyces cerevisiae

Saccharomyces cerevisiae septin mutants have pleiotropic defects, which include the formation of abnormally elongated buds. This bud morphology results at least in part from a cell cycle delay imposed by the Cdc28p-inhibitory kinase Swe1p. Mutations in three other genes (GIN4, encoding a kinase related to the Schizosaccharomyces pombe mitotic inducer Nim1p; CLA4, encoding a p21-activated kinase; and NAP1, encoding a Clb2p-interacting protein) also produce perturbations of septin organization associated with an Swe1p-dependent cell cycle delay. The effects of gin4, cla4, and nap1 mutations are additive, indicating that these proteins promote normal septin organization through pathways that are at least partially independent. In contrast, mutations affecting the other two Nim1p-related kinases in S. cerevisiae, Hsl1p and Kcc4p, produce no detectable effect on septin organization. However, deletion of HSL1, but not of KCC4, did produce a cell cycle delay under some conditions; this delay appears to reflect a direct role of Hsl1p in the regulation of Swe1p. As shown previously, Swe1p plays a central role in the morphogenesis checkpoint that delays the cell cycle in response to defects in bud formation. Swe1p is localized to the nucleus and to the daughter side of the mother bud neck prior to its degradation in G(2)/M phase. Both the neck localization of Swe1p and its degradation require Hsl1p and its binding partner Hsl7p, both of which colocalize with Swe1p at the daughter side of the neck. This localization is lost in mutants with perturbed septin organization, suggesting that the release of Hsl1p and Hsl7p from the neck may reduce their ability to inactivate Swe1p and thus contribute to the G(2) delay observed in such mutants. In contrast, treatments that perturb actin organization have little effect on Hsl1p and Hsl7p localization, suggesting that such treatments must stabilize Swe1p by another mechanism. The apparent dependence of Swe1p degradation on localization of the Hsl1p-Hsl7p-Swe1p module to a site that exists only in budded cells may constitute a mechanism for deactivating the morphogenesis checkpoint when it is no longer needed (i.e., after a bud has formed).     

Zds2p regulates Swe1p-dependent polarized cell growth in Saccharomyces cerevisiae via a novel Cdc55p interaction domain

Deletion of the paralogs ZDS1 and ZDS2 in the budding yeast Saccharomyces cerevisiae causes a mis-regulation of polarized cell growth. Here we show a function for these genes as regulators of the Swe1p (Wee1p) kinase-dependent G2/M checkpoint. We identified a conserved domain in the C-terminus of Zds2p consisting of amino acids 813-912 (hereafter referred to as ZH4 for Zds homology 4) that is required for regulation of Swe1p-dependent polarized bud growth. ZH4 is shown by protein affinity assays to be necessary and sufficient for interaction with Cdc55p, a regulatory subunit of protein phosphatase 2A (PP2A). We hypothesized that the Zds proteins are in a pathway that negatively regulates the Swe1p-dependent G2/M checkpoint via Cdc55p. Supporting this model, deletion of CDC55 rescues the aberrant bud morphology of a zds1Δzds2Δ strain. We also show that expression of ZDS1 or ZDS2 from a strong galactose-inducible promoter can induce mitosis even when the Swe1p-dependent G2/M checkpoint is activated by mis-organization of the actin cytoskeleton. This negative regulation requires the CDC55 gene. Together these data indicate that the Cdc55p/Zds2p module has a function in the regulation of the Swe1p-dependent G2/M checkpoint.