New regulators of a high affinity Ca2+ influx system revealed through a genome-wide screen in yeast

The bakers' yeast Saccharomyces cerevisiae utilizes a high affinity Ca(2+) influx system (HACS) to survive assaults by mating pheromones, tunicamycin, and azole-class antifungal agents. HACS consists of two known subunits, Cch1 and Mid1, that are homologous and analogous to the catalytic α-subunits and regulatory α2δ-subunits of mammalian voltage-gated calcium channels, respectively. To search for additional subunits and regulators of HACS, a collection of gene knock-out mutants was screened for abnormal uptake of Ca(2+) after exposure to mating pheromone or to tunicamycin. The screen revealed that Ecm7 is required for HACS function in most conditions. Cycloheximide chase experiments showed that Ecm7 was stabilized by Mid1, and Mid1 was stabilized by Cch1 in non-signaling conditions, suggesting they all interact. Ecm7 is a member of the PMP-22/EMP/MP20/Claudin superfamily of transmembrane proteins that includes γ-subunits of voltage-gated calcium channels. Eleven additional members of this superfamily were identified in yeast, but none was required for HACS activity in response to the stimuli. Remarkably, many dozens of genes involved in vesicle-mediated trafficking and protein secretion were required to prevent spontaneous activation of HACS. Taken together, the findings suggest that HACS and calcineurin monitor performance of the membrane trafficking system in yeasts and coordinate compensatory processes. Conservation of this quality control system in Candida glabrata suggests that many pathogenic species of fungi may utilize HACS and calcineurin to resist azoles and other compounds that target membrane biosynthesis.     

Fig1p facilitates Ca2+ influx and cell fusion during mating of Saccharomyces cerevisiae

During the mating process of yeast cells, two Ca2+ influx pathways become activated. The resulting elevation of cytosolic free Ca2+ activates downstream signaling factors that promote long term survival of unmated cells, but the roles of Ca2+ in conjugation have not been described. The high affinity Ca2+ influx system is composed of Cch1p and Mid1p and sensitive to feedback inhibition by calcineurin, a Ca2+/calmodulin-dependent protein phosphatase. To identify components and regulators of the low affinity Ca2+ influx system (LACS), we screened a collection of pheromone-responsive genes that when deleted lead to defects in LACS activity but not high affinity Ca2+ influx system activity. Numerous factors implicated in polarized morphogenesis and cell fusion (Fus1p, Fus2p, Rvs161p, Bni1p, Spa2p, and Pea2p) were found to be necessary for LACS activity. Each of these factors was also required for activation of the cell integrity mitogen-activated protein kinase cascade during the response to alpha-factor. Interestingly a polytopic plasma membrane protein, Fig1p, was required for LACS activity but not required for activation of Mpk1p mitogen-activated protein kinase. Mpk1p was not required for LACS activity, suggesting Mpk1p and Fig1p define two independent branches in the pheromone response pathways. Fig1p-deficient mutants exhibit defects in the cell-cell fusion step of mating, but unlike other fus1 and fus2 mutants the fusion defect of fig1 mutants can be largely suppressed by high Ca2+ conditions, which bypass the requirement for LACS. These findings suggest Fig1p is an important component or regulator of LACS and provide the first evidence for a role of Ca2+ signals in the cell fusion step of mating.     

Essential role of calcineurin in response to endoplasmic reticulum stress

Depletion of calcium ions (Ca2+) from the endoplasmic reticulum (ER) of yeast cells resulted in the activation of the unfolded protein response (UPR) signaling pathway involving Ire1p and Hac1p. The depleted ER also stimulated Ca2+ influx at the plasma membrane through the Cch1p-Mid1p Ca2+ channel and another system. Surprisingly, both Ca2+ influx systems were stimulated upon accumulation of misfolded proteins in the ER even in the presence of Ca2+. The ability of misfolded ER proteins to stimulate Ca2+ influx at the plasma membrane did not require Ire1p or Hac1p, and Ca2+ influx and signaling factors were not required for initial UPR signaling. However, activation of the Ca2+ channel, calmodulin, calcineurin and other factors was necessary for long-term survival of cells undergoing ER stress. A similar calcium cell survival (CCS) pathway operates in the pathogenic fungi and promotes resistance to azole antifungal drugs. These findings reveal an unanticipated new regulatory mechanism that couples ER stress to Ca2+ influx and signaling pathways, which help to prevent cell death and promote resistance to an important class of fungistatic drugs.     

A homolog of voltage-gated Ca(2+) channels stimulated by depletion of secretory Ca(2+) in yeast

In animal cells, capacitative calcium entry (CCE) mechanisms become activated specifically in response to depletion of calcium ions (Ca(2+)) from secretory organelles. CCE serves to replenish those organelles and to enhance signaling pathways that respond to elevated free Ca(2+) concentrations in the cytoplasm. The mechanism of CCE regulation is not understood because few of its essential components have been identified. We show here for the first time that the budding yeast Saccharomyces cerevisiae employs a CCE-like mechanism to refill Ca(2+) stores within the secretory pathway. Mutants lacking Pmr1p, a conserved Ca(2+) pump in the secretory pathway, exhibit higher rates of Ca(2+) influx relative to wild-type cells due to the stimulation of a high-affinity Ca(2+) uptake system. Stimulation of this Ca(2+) uptake system was blocked in pmr1 mutants by expression of mammalian SERCA pumps. The high-affinity Ca(2+) uptake system was also stimulated in wild-type cells overexpressing vacuolar Ca(2+) transporters that competed with Pmr1p for substrate. A screen for yeast mutants specifically defective in the high-affinity Ca(2+) uptake system revealed two genes, CCH1 and MID1, previously implicated in Ca(2+) influx in response to mating pheromones. Cch1p and Mid1p were localized to the plasma membrane, coimmunoprecipitated from solubilized membranes, and shown to function together within a single pathway that ensures that adequate levels of Ca(2+) are supplied to Pmr1p to sustain secretion and growth. Expression of Cch1p and Mid1p was not affected in pmr1 mutants. The evidence supports the hypothesis that yeast maintains a homeostatic mechanism related to CCE in mammalian cells. The homology between Cch1p and the catalytic subunit of voltage-gated Ca(2+) channels raises the possibility that in some circumstances CCE in animal cells may involve homologs of Cch1p and a conserved regulatory mechanism.     

The Saccharomyces cerevisiae Ca2+ channel Cch1pMid1p is essential for tolerance to cold stress and iron toxicity

Cch1p and Mid1p are components of a high-affinity Ca(2+)-permeable channel in the yeast plasma membrane. Here, we show that growth of mutants in the Cch1pMid1p channel is markedly hypersensitive to low temperature and to high iron concentration in the medium. Both phenotypes were suppressed by high Ca(2+) concentration. Iron stress elicited an increased Ca(2+) influx into both wild type and cch1Deltamid1Delta yeast. Inhibition of calcineurin strongly depressed growth of iron-stressed wild type yeast, indicating that calcineurin is a downstream element of the iron stress response. Iron hypersensitivity of the cch1Deltamid1Delta mutant was not associated with an increased iron uptake. An involvement of oxidative stress in the iron-hypersensitive phenotype was indicated by the findings that the antioxidants tocopheryl acetate and (ethyl)glutathione improved growth and viability of the iron-stressed mutant. Further, the degree of glutathione oxidation was increased in the presence of iron. The results indicate that iron stress leads to an increased oxidative poise and that Cch1pMid1p is essential to tolerate this condition.     

Role of Fig1, a component of the low-affinity calcium uptake system, in growth and sexual development of filamentous fungi

The function of Fig1, a transmembrane protein of the low-affinity calcium uptake system (LACS) in fungi, was examined for its role in the growth and development of the plant pathogen Fusarium graminearum. The Δfig1 mutants failed to produce mature perithecia, and sexual development was halted prior to the formation of perithecium initials. The loss of Fig1 function also resulted in a reduced vegetative growth rate. Macroconidium production was reduced 70-fold in the Δfig1 mutants compared to the wild type. The function of the high-affinity calcium uptake system (HACS), comprised of the Ca(2+) channels Mid1 and Cch1, was previously characterized for F. graminearum. To better understand the roles of the LACS and the HACS, Δfig1 Δmid1, Δfig1 Δcch1, and Δfig1 Δmid1 Δcch1 double and triple mutants were generated, and the phenotypes of these mutants were more severe than those of the Δfig1 mutants. Pathogenicity on wheat was unaffected for the Δfig1 mutants, but the Δfig1 Δmid1, Δfig1 Δcch1, and Δfig1 Δmid1 Δcch1 mutants, lacking both LACS and HACS functions, had reduced pathogenicity. Additionally, Δfig1 mutants of Neurospora crassa were examined and did not affect filamentous growth or female fertility in a Δfig1 mating type A strain, but the Δfig1 mating type a strain failed to produce fertile fruiting bodies. These results are the first report of Fig1 function in filamentous ascomycetes and expand its role to include complex fruiting body and ascus development.     

A homolog of mammalian, voltage-gated calcium channels mediates yeast pheromone-stimulated Ca2+ uptake and exacerbates the cdc1(Ts) growth defect.

Previous studies attributed the yeast (Saccharomyces cerevisiae) cdc1(Ts) growth defect to loss of an Mn2+-dependent function. In this report we show that cdc1(Ts) temperature-sensitive growth is also associated with an increase in cytosolic Ca2+. We identified two recessive suppressors of the cdc1(Ts) temperature-sensitive growth which block Ca2+ uptake and accumulation, suggesting that cytosolic Ca2+ exacerbates or is responsible for the cdc1(Ts) growth defect. One of the cdc1(Ts) suppressors is identical to a gene, MID1, recently implicated in mating pheromone-stimulated Ca2+ uptake. The gene (CCH1) corresponding to the second suppressor encodes a protein that bears significant sequence similarity to the pore-forming subunit (alpha1) of plasma membrane, voltage-gated Ca2+ channels from higher eukaryotes. Strains lacking Mid1 or Cch1 protein exhibit a defect in pheromone-induced Ca2+ uptake and consequently lose viability upon mating arrest. The mid1delta and cch1delta mutants also display reduced tolerance to monovalent cations such as Li+, suggesting a role for Ca2+ uptake in the calcineurin-dependent ion stress response. Finally, mid1delta cch1delta double mutants are, by both physiological and genetic criteria, identical to single mutants. These and other results suggest Mid1 and Cch1 are components of a yeast Ca2+ channel that may mediate Ca2+ uptake in response to mating pheromone, salt stress, and Mn2+ depletion.     

Calcium influx and signaling in yeast stimulated by intracellular sphingosine 1-phosphate accumulation

In mammalian cells, intracellular sphingosine 1-phosphate (S1P) can stimulate calcium release from intracellular organelles, resulting in the activation of downstream signaling pathways. The budding yeast Saccharomyces cerevisiae expresses enzymes that can synthesize and degrade S1P and related molecules, but their possible role in calcium signaling has not yet been tested. Here we examine the effects of S1P accumulation on calcium signaling using a variety of yeast mutants. Treatment of yeast cells with exogenous sphingosine stimulated Ca(2+) accumulation through two distinct pathways. The first pathway required the Cch1p and Mid1p subunits of a Ca(2+) influx channel, depended upon the function of sphingosine kinases (Lcb4p and Lcb5p), and was inhibited by the functions of S1P lyase (Dpl1p) and the S1P phosphatase (Lcb3p). The biologically inactive stereoisomer of sphingosine did not activate this Ca(2+) influx pathway, suggesting that the active S1P isomer specifically stimulates a calcium-signaling mechanism in yeast. The second Ca(2+) influx pathway stimulated by the addition of sphingosine was not stereospecific, was not dependent on the sphingosine kinases, occurred only at higher doses of added sphingosine, and therefore was likely to be nonspecific. Mutants lacking both S1P lyase and phosphatase (dpl1 lcb3 double mutants) exhibited constitutively high Ca(2+) accumulation and signaling in the absence of added sphingosine, and these effects were dependent on the sphingosine kinases. These results show that endogenous S1P-related molecules can also trigger Ca(2+) accumulation and signaling. Several stimuli previously shown to evoke calcium signaling in wild-type cells were examined in lcb4 lcb5 double mutants. All of the stimuli produced calcium signals independent of sphingosine kinase activity, suggesting that phosphorylated sphingoid bases might serve as messengers of calcium signaling in yeast during an unknown cellular response.     

An essential role of the yeast pheromone-induced Ca2+ signal is to activate calcineurin.

Previous studies showed that, in wild-type (MATa) cells, alpha-factor causes an essential rise in cytosolic Ca2+. We show that calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, is one target of this Ca2+ signal. Calcineurin mutants lose viability when incubated with mating pheromone, and overproduction of constitutively active (Ca(2+)-independent) calcineurin improves the viability of wild-type cells exposed to pheromone in Ca(2+)-deficient medium. Thus, one essential consequence of the pheromone-induced rise in cytosolic Ca2+ is activation of calcineurin. Although calcineurin inhibits intracellular Ca2+ sequestration in yeast cells, neither increased extracellular Ca2+ nor defects in vacuolar Ca2+ transport bypasses the requirement for calcineurin during the pheromone response. These observations suggest that the essential function of calcineurin in the pheromone response may be distinct from its modulation of intracellular Ca2+ levels. Mutants that do not undergo pheromone-induced cell cycle arrest (fus3, far1) show decreased dependence on calcineurin during treatment with pheromone. Thus, calcineurin is essential in yeast cells during prolonged exposure to pheromone and especially under conditions of pheromone-induced growth arrest. Ultrastructural examination of pheromone-treated cells indicates that vacuolar morphology is abnormal in calcineurin-deficient cells, suggesting that calcineurin may be required for maintenance of proper vacuolar structure or function during the pheromone response.     

Identification of functional domains of Mid1, a stretch-activated channel component, necessary for localization to the plasma membrane and Ca2+ permeation

The Saccharomyces cerevisiae MID1 gene product (Mid1) is a stretch-activated Ca(2+)-permeable channel component required for Ca2+ influx and the maintenance of viability of cells exposed to the mating pheromone, alpha-factor. It is composed of 548-amino-acid (aa) residues with four hydrophobic segments, H1 (aa 2-22), H2 (aa 92-111), H3 (aa 337-356) and H4 (aa 366-388). It also has 16 putative N-glycosylation sites. In this study, sequentially truncated Mid1 proteins conjugated with GFP were expressed in S. cerevisiae cells. The truncated protein containing the region from H1 to H3 (Mid1(1-360)-GFP) localized normally in the plasma and endoplasmic reticulum (ER) membranes and complemented the low viability and Ca(2+)-uptake activity of the mid1 mutant, whereas Mid1(1-133)-GFP containing the region from H1 to H2 did not. Mid1(Delta3-22)-GFP lacking the H1 region failed to localize in the plasma membrane. Membrane fractionation showed that Mid1(1-22)-GFP containing only H1 localized in the plasma membrane in the presence of alpha-factor, suggesting that H1 is a signal sequence responsible for the alpha-factor-induced Mid1 delivery to the plasma membrane. The region from H1 to H3 is required for the localization of Mid1 in the plasma and ER membranes. Finally, trafficking of Mid1-GFP to the plasma membrane was dependent on the N-glycosylation of Mid1 and the transporter protein Sec12.     

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.     

酿酒酵母细胞中钙离子信号传导途径的研究进展

酿酒酵母（SaccharomycescP增v括fdP）细胞可以通过ca2＋／钙调磷酸酶信号途径来应对许多外界环境胁迫。在交配信息素、盐或者其他环境压力存在的条件下,钙离子会通过细胞质膜上的未鉴定的钙转运蛋白x和M或者由Cchl和Midl组成的钙通道进入细胞质。胞质内钙离子浓度的增加会激活细胞质里的钙调磷酸酶（calcineurin）。钙调磷酸酶的一个非常重要的作用是去磷酸化细胞质内的转录因子Crzl,造成它快速地从细胞质转移到细胞核,从而诱导包括液泡膜上钙泵蛋白基因PMCl以及内质网膜和高尔基体膜上钙泵蛋白基因尸脚,在内的目标基因的表达。这两个钙泵蛋白和液泡膜上的Ca2＋／H＋交换蛋白Vcxl一起作用,将细胞质内的钙离子浓度控制在50～200nmol／L的正常生理浓度内．使细胞能够正常生长。该综述主要论述了酿酒酵母细胞内Ca2＋／钙调磷酸酶信号途径的最新研究进展。     

Subcellular localization and oligomeric structure of the yeast putative stretch-activated Ca2+ channel component Mid1

The yeast Mid1 protein with an apparent molecular mass of 100 kDa is required for Ca2+ influx stimulated by the mating pheromone and by a capacitative calcium entrylike mechanism acting in response to Ca2+ depletion from the endoplasmic reticulum (ER) and functions as a stretch-activated Ca2+ -permeable channel when expressed in mammalian cells. Our previous work with protease protection experiments has indicated that Mid1 is present in the plasma membrane. In this study, we examined a possible intracellular localization of this protein by indirect fluorescence microscopy and found that Mid1 is present in the ER membrane as well as the plasma membrane. Intracellular fluorescence images for Mid1 were the same as those for the ER marker protein Sec71 but quite different from those of the Golgi protein Ypt1. The results were confirmed by membrane fractionation using Angiografin density gradient analysis. We also investigated the oligomeric structures and protein levels of Mid1 and found that Mid1 forms a 200-kDa oligomer by disulfide bonding. The protein level and modification of Mid1 in the plasma membrane and the ER membrane were unchanged by the mating pheromone. These findings provide new insight into the function of Mid1 in relation to localization, modification, and activation mechanisms.     

Localized feedback phosphorylation of Ste5p scaffold by associated MAPK cascade

Scaffold proteins play pivotal roles during signal transduction. In Saccharomyces cerevisiae, the Ste5p scaffold protein is required for activation of the mating MAPK cascade in response to mating pheromone and assembles a G protein-MAPK cascade complex at the plasma membrane. To serve this function, Ste5p undergoes a regulated localization event involving nuclear shuttling and recruitment to the cell cortex. Here, we show that Ste5p is also subject to two types of phosphorylation and increases in abundance as a result of MAPK activation. During vegetative growth, Ste5p is basally phosphorylated through a process regulated by the CDK Cdc28p. During mating pheromone signaling, Ste5p undergoes increased phosphorylation by the mating MAPK cascade. Multiple kinases of the mating MAPK cascade contribute to pheromone-induced phosphorylation of Ste5p, with the mating MAPKs contributing the most. Pheromone induction or overexpression of the Ste4p Gbeta subunit increases the abundance of Ste5p at a post-translational step, as long as the mating MAPKs are present. Increasing the level of MAPK activation increases the amount of Ste5p at the cell cortex. Analysis of Ste5p localization mutants reveals a strict requirement for Ste5p recruitment to the plasma membrane for the pheromone-induced phosphorylation. These results suggest that the pool of Ste5p that is recruited to the plasma membrane selectively undergoes feedback phosphorylation by the associated MAPKs, leading to an increased pool of Ste5p at the site of polarized growth. These findings provide evidence of a spatially regulated mechanism for post-activation control of a signaling scaffold that potentiates pathway activation.     

Activation of an Essential Calcium Signaling Pathway in Saccharomyces cerevisiae by Kch1 and Kch2, Putative Low-Affinity Potassium Transporters

In the budding yeast Saccharomyces cerevisiae, mating pheromones activate a high-affinity Ca²⁺ influx system (HACS) that activates calcineurin and is essential for cell survival. Here we identify extracellular K⁺ and a homologous pair of transmembrane proteins, Kch1 and Kch2 (Prm6), as necessary components of the HACS activation mechanism. Expression of Kch1 and especially Kch2 was strongly induced during the response to mating pheromones. When forcibly overexpressed, Kch1 and Kch2 localized to the plasma membrane and activated HACS in a fashion that depended on extracellular K⁺ but not pheromones. They also promoted growth of trk1 trk2 mutant cells in low K⁺ environments, suggesting they promote K⁺ uptake. Voltage-clamp recordings of protoplasts revealed diminished inward K⁺ currents in kch1 kch2 double-mutant cells relative to the wild type. Conversely, heterologous expression of Kch1 in HEK293T cells caused the appearance of inwardly rectifying K⁺ currents. Collectively, these findings suggest that Kch1 and Kch2 directly promote K⁺ influx and that HACS may electrochemically respond to K⁺ influx in much the same way as the homologous voltage-gated Ca²⁺ channels in most animal cell types.     

Simulating calcium influx and free calcium concentrations in yeast

Yeast can proliferate in environments containing very high Ca(2+) primarily due to the activity of vacuolar Ca(2+) transporters Pmc1 and Vcx1. Yeast mutants lacking these transporters fail to grow in high Ca(2+) environments, but growth can be restored by small increases in environmental Mg(2+). Low extracellular Mg(2+) appeared to competitively inhibit novel Ca(2+) influx pathways and to diminish the concentration of free Ca(2+) in the cytoplasm, as judged from the luminescence of the photoprotein aequorin. These Mg(2+)-sensitive Ca(2+) influx pathways persisted in yvc1 cch1 double mutants. Based on mathematical models of the aequorin luminescence traces, we propose the existence in yeast of at least two Ca(2+) transporters that undergo rapid feedback inhibition in response to elevated cytosolic free Ca(2+) concentration. Finally, we show that Vcx1 helps return cytosolic Ca(2+) toward resting levels after shock with high extracellular Ca(2+) much more effectively than Pmc1 and that calcineurin, a protein phosphatase regulator of Vcx1 and Pmc1, had no detectable effects on these factors within the first few minutes of its activation. Therefore, computational modeling of Ca(2+) transport and signaling in yeast can provide important insights into the dynamics of this complex system.