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.     

Characterization of the energy-dependent, mating factor-activated Ca influx in Saccharomyces cerevisiae

The yeast mating pheromones, a and alpha factors, bind to specific G protein-coupled receptors in haploid cells and bring about both growth arrest in the early G1 phase of the cell cycle and differentiation into mating capable cells. This induces an increase in Ca2+ influx leading to elevated intracellular calcium concentrations, which has been shown to be essential for subsequent downstream events and the mating process itself [1]. We have characterized the alpha factor induced increase in cellular Ca2+ in wild type S. cerevisiae and in the temperature-sensitive cell division cycle mutants cdc7 and cdc28 which are growth-arrested at the G0-G1 border at the nonpermissive temperature. We observed a 2-4 fold increase in the initial velocity of Ca2+ influx in alpha factor-treated wild-type cells and in cdc7 and cdc28 cells grown at the nonpermissive temperature. Calcium influx was energy dependent, inhibited by membrane depolarization and slightly increased by hyperpolarization. Furthermore, Ca2+ influx was sensitive to both divalent and trivalent cations, but was unaffected by nifedipine and verapamil. These data demonstrate that budding yeast possesses a regulated Ca2+ transport mechanism, the activation of which is dependent upon exit out of the cell cycle and growth cessation. This transport mechanism has many similarities to that observed in mitogen-stimulated mammalian cells.     

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.     

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.     

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.     

Phe356 in the yeast Ca2+ channel component Mid1 is a key residue for viability after exposure to alpha-factor

The yeast Mid1 protein is an integral membrane protein required for the viability of differentiated cells and Ca2+ influx induced by mating pheromone. Our previous study has identified a loss-of-function mutation, F356S. The F356S mutant is completely unable to maintain viability, but still has Ca2+ accumulation activity near the wild-type level. Here we further examined in detail the F356S mutation to unravel the function of Phe356. After exposure to the pheromone, the F356S mutant was not fully rescued by high extracellular Ca2+, like the mid1 null mutant, suggesting that Phe356 and Mid1 itself are also required for viability maintenance mechanism that does not involve Ca2+ signalling. Substitutions of hydrophilic amino acids for Phe356 caused lethality and low Ca2+ accumulation, but those of hydrophobic amino acids did not. Substitutions of small amino acids for Phe356 caused a significantly reduced viability, but did not affect Ca2+ accumulation. We suggest that the hydrophobicity of the Phe356 residue is important for both viability maintenance and Ca2+ uptake, and that its size for viability maintenance.     

Phospholipase D1 is required for efficient mating projection formation in Saccharomyces cerevisiae

Phospholipase D1 (PLD1) is an important enzyme involved in lipid signal transduction in eukaryotes. A role for PLD1 in signaling in Saccharomyces cerevisiae was examined. Pheromone response in yeast is controlled by a well-characterized protein kinase cascade. Loss of PLD1 activity was found to impair pheromone-induced changes in cellular morphology that result in formation of mating projections. The rate at which projections appeared following pheromone treatment was delayed, suggesting that PLD1 facilitates the execution of a rate-limiting step in morphogenesis. Mutants were found to be less sensitive to pheromone, again arguing that PLD1 is acting at a rate-limiting step. The fact that morphogenesis is most dramatically affected indicates that PLD1 functions primarily in the morphogenic branch of the pheromone response pathway.     

Transient increase of Ca2+ uptake as a signal for mating pheromone-induced differentiation in the heterobasidiomycetous yeast Rhodosporidium toruloides.

The role of Ca2+ for the signaling of rhodotorucine A, a mating pheromone of Rhodosporidium toruloides, was investigated. The efficiency with which the target cells responded to the mating pheromone was dependent on the Ca2+ concentration in the medium. The pheromone induced a very rapid and transient increase of Ca2+ uptake in the recipient cell. We concluded that the transient increase in the intracellular Ca2+ concentration could play an essential role in the control of differentiation by the pheromone.     

MID1, a novel Saccharomyces cerevisiae gene encoding a plasma membrane protein,is required for Ca2+ influx and mating.

By establishing a unique screening method, we have isolated yeast mutants that die only after differentiating into cells with a mating projection, and some of them are also defective in Ca2+ signaling. The mutants were classified into five complementation groups, one of which we studied extensively. This mutation defines a new gene, designated MID1, which encodes an N-glycosylated, integral plasma membrane protein with 548 amino acid residues. The mid1-1 mutant has low Ca2+ uptake activity, loses viability after receiving mating pheromones, and escapes death when incubated with high concentrations of CaCl2. The MID1 gene is nonessential for vegetative growth. The efficiency of mating between MATa mid1-1 and MAT alpha mid1-1 cells is low. These results demonstrate that MID1 is required for Ca2+ influx and mating.     

Lymphocyte adhesion-dependent calcium signaling in human endothelial cells

Vascular endothelial cells (ECs) can undergo dramatic phenotypic and functional alterations in response to humoral and cellular stimuli. These changes promote endothelial participation in the inflammatory response through active recruitment of immune effector cells, increased vascular permeability, and alteration in vascular tone. In an attempt to define early events in lymphocyte-mediated EC signaling, we investigated cytosolic-free calcium (Ca2+) changes in single, Fluo-3- labeled human umbilical vein ECs (HUVECs), using an ACAS interactive laser cytometer. Of all lymphocyte subsets tested, allogeneic CD3-, CD56+ natural killer (NK) cells uniquely elicited oscillatory EC Ca2+ signals in cytokine (interleukin [IL]-1- or tumor necrosis factor [TNF])-treated ECs. The induction of these signals required avid intercellular adhesion, consisted of both Ca2+ mobilization and extracellular influx, and was associated with EC inositol phosphate (IP) generation. Simultaneous recording of NK and EC Ca2+ signals using two-color fluorescence detection revealed that, upon adhesion, NK cells flux prior to EC. Lymphocyte Ca2+ buffering with 1,2-bis-5-methyl-amino- phenoxylethane-N,N,N'-tetra-acetoxymethyl acetate (MAPTAM) demonstrated that lymphocyte fluxes are, in fact, prerequisites for the adhesion- dependent EC signals. mAb studies indicate that the beta 2 integrin- intercellular adhesion molecule (ICAM)-1 adhesion pathway is critically involved. However, ICAM-1 antisense oligonucleotide inhibition of IL-1- mediated ICAM-1 hyperinduction had no effect on EC Ca2+ signaling in lymphocyte-EC conjugates, indicating that additional cytokine-induced EC alteration is required. These experiments combine features of lymphocyte-endothelial interactions, intercellular adhesion, EC cytokine activation and transmembrane signaling. The results implicate the IP/Ca2+ second messenger pathway in EC outside-in signaling induced by cytotoxic lymphocytes, and suggest that these signals may play a role in EC alteration by lymphocyte adhesion.     

Checkpoints in a yeast differentiation pathway coordinate signaling during hyperosmotic stress

All eukaryotes have the ability to detect and respond to environmental and hormonal signals. In many cases these signals evoke cellular changes that are incompatible and must therefore be orchestrated by the responding cell. In the yeast Saccharomyces cerevisiae, hyperosmotic stress and mating pheromones initiate signaling cascades that each terminate with a MAP kinase, Hog1 and Fus3, respectively. Despite sharing components, these pathways are initiated by distinct inputs and produce distinct cellular behaviors. To understand how these responses are coordinated, we monitored the pheromone response during hyperosmotic conditions. We show that hyperosmotic stress limits pheromone signaling in at least three ways. First, stress delays the expression of pheromone-induced genes. Second, stress promotes the phosphorylation of a protein kinase, Rck2, and thereby inhibits pheromone-induced protein translation. Third, stress promotes the phosphorylation of a shared pathway component, Ste50, and thereby dampens pheromone-induced MAPK activation. Whereas all three mechanisms are dependent on an increase in osmolarity, only the phosphorylation events require Hog1. These findings reveal how an environmental stress signal is able to postpone responsiveness to a competing differentiation signal, by acting on multiple pathway components, in a coordinated manner.     

Ca2+ signaling by distinct endothelin peptides in glomerular mesangial cells.

Ca2+ signaling by peptides of the endothelin (ET) gene family was studied in cultured glomerular mesangial cells. In addition to the increase in cytosolic free [Ca2+] ([Ca2+]i) previously described for ET-1, we also observed that ET-2, ET-3, and sarafotoxin S6b generate similar [Ca2+]i waveforms but with dissimilar potencies and kinetics. The prepro form of ET-1 was inactive, suggesting that mature ET peptides are constrained in an inactive conformation within the preproET species. ET isopeptides caused both release of Ca2+ from intracellular stores and Ca2+ influx via a voltage- and dihydropyridine-insensitive pathway. ET-mediated Ca2+ influx was independent of the increase in [Ca2+]i. Activation of protein kinase C inhibited ET-induced Ca2+ signaling, whereas addition of ET to protein kinase C-depleted cells resulted in enhanced [Ca2+]i waveforms. Mesangial cells also demonstrated a marked adaptive desensitization response to ET. These data demonstrate that Ca2+ signaling is a common response to different ET peptides in glomerular mesangial cells and that activation of protein kinase C down-regulates these Ca2+ signals.     

Pheromone-induced phosphorylation of a G protein beta subunit in S. cerevisiae is associated with an adaptive response to mating pheromone

The mating pheromone response in S. cerevisiae is activated by a G protein-mediated signaling pathway in which G beta gamma is the active transducer of the signal. When exogenous pheromone is added to vegetatively growing cells, G beta is rapidly phosphorylated at several sites; phosphorylation does not require de novo protein synthesis. A mutation in G beta was constructed that eliminates signal-induced phosphorylation. This mutation leads to enhanced sensitivity to and impaired ability to recover from pheromone, but does not affect the ability of G beta gamma to transmit the mating signal. These phenotypes suggest that G protein phosphorylation mediates an adaptive response to pheromone-induced signaling. G beta phosphorylation does not require either the pheromone receptor C-terminus or the product of the SST2 gene, both of which mediate separate adaptive responses to pheromone. However, G beta phosphorylation is greatly facilitated by the presence of the G alpha subunit, which has also been shown to participate in an adaptation to pheromone.     

The Leu-132 of the Ste4(Gbeta) subunit is essential for proper coupling of the G protein with the Ste2 alpha factor receptor during the mating pheromone response in yeast

In order to identify amino acid residues of Ste4p involved in receptor recognition and/or receptor-G protein coupling, we employed random in vitro mutagenesis and a genetic screening to isolate mutant Ste4p subunits with altered pheromone response. We generated a plasmid library containing randomly mutagenized Ste4 ORFs, followed by phenotypic selection of ste4p mutants by altered alpha pheromone response in yeast cells. Subsequently, we analyzed mutant ste4-10 which has a replacement of the almost universally conserved leucine 132 by phenylalanine. This residue lies in the first blade of the beta propeller structure proposed by crystallographic analysis. By overexpression experiments we found that mutant ste4p subunit triggers the mating pathway at wild type levels in both wild type and receptorless strains. When expressed in a ste4 background, however, the mutant G protein is activated inefficiently by mating pheromone in both a and alpha cells. The mutant ste4-10p was tested in the two-hybrid system and found to be defective in its interaction with the Gpa1p, but has a normal association with the C-termini end of the Ste2p receptor. These observations strongly suggest that the Leu-132 of the Ste4p subunit is essential for efficient activation of the G protein by the pheromone-stimulated receptor and that this domain could be an important point for physical interaction between the Gbeta and the Galpha subunits.     

A walk-through of the yeast mating pheromone response pathway

The intracellular signal transduction pathway by which the yeast Saccharomyces cerevisiae responds to the presence of peptide mating pheromone in its surroundings is one of the best understood signaling pathways in eukaryotes, yet continues to generate new surprises and insights. In this review, we take a brief walk down the pathway, focusing on how the signal is transmitted from the cell-surface receptor-coupled G protein, via a MAP kinase cascade, to the nucleus.