Prm1 prevents contact-dependent lysis of yeast mating pairs

Membrane fusion requires localized destabilization of two phospholipid bilayers, but unrestrained membrane destabilization could result in lysis. prm1 mutant yeast cells have a defect at the plasma membrane fusion stage of mating that typically results in the accumulation of prezygotes that have fingers of membrane-bound cytoplasm projecting from one cell of each pair into its mating partner in the direction of the osmotic gradient between the cells. However, some prm1 mating pairs fuse successfully whereas the two cells in other prm1 mating pairs simultaneously lyse. Lysis only occurs if both mating partners are prm1 mutants. Osmotic stabilization does not protect prm1 mating pairs from lysis, indicating that lysis is not caused by a cell wall defect. prm1 mating pairs without functional mitochondria still lyse, ruling out programmed cell death. No excess lysis was found after pheromone treatment of haploid prm1 cells, and lysis did not occur in mating pairs when prm1 was combined with the fus1 and fus2 mutations to block cell wall remodeling. Furthermore, short (<1 microm) cytoplasmic microfingers indicating the completion of cell wall remodeling appeared immediately before lysis. In combination, these results demonstrate that plasma membrane contact is a prerequisite for lysis. Cytoplasmic microfingers are unlikely to cause lysis since most prm1 mating pairs with microfingers do not lyse, and microfingers were also detected before fusion in some wild-type mating pairs. The lysis of prm1 mutant mating pairs suggests that the Prm1 protein stabilizes the membrane fusion event of yeast mating.     

Genetic Analysis of Default Mating Behavior in Saccharomyces Cerevisiae

Haploid Saccharomyces cerevisiae cells find each other during conjugation by orienting their growth toward each other along pheromone gradients (chemotropism). However, when their receptors are saturated for pheromone binding, yeast cells must select a mate by executing a default pathway in which they choose a mating partner at random. We previously demonstrated that this default pathway requires the SPA2 gene. In this report we show that the default mating pathway also requires the AXL1, FUS1, FUS2, FUS3, PEA2, RVS161, and BNI1 genes. These genes, including SPA2, are also important for efficient cell fusion during chemotropic mating. Cells containing null mutations in these genes display defects in cell fusion that subtly affect mating efficiency. In addition, we found that the defect in default mating caused by mutations in SPA2 is partially suppressed by multiple copies of two genes, FUS2 and MFA2. These findings uncover a molecular relationship between default mating and cell fusion. Moreover, because axl1 mutants secrete reduced levels of a-factor and are defective at both cell fusion and default mating, these results reveal an important role for a-factor in cell fusion and default mating. We suggest that default mating places a more stringent requirement on some aspects of cell fusion than does chemotropic mating.     

Mutants of Saccharomyces cerevisiae unresponsive to cell division control by polypeptide mating hormone

Temperature-sensitive mutations that produce insensitivity to division arrest by alpha-factor, a mating pheromone, were isolated in an MATa strain of Saccharomyces cerevisiae and shown by complementation studies to difine eight genes. All of these mutations (designated ste) produce sterility at the restrictive temperature in MATa cells, and mutations in seven of the genes produce sterility in MAT alpha cells. In no case was the sterility associated with these mutations coorectible by including wild-type cells of the same mating type in the mating test nor did nay of the mutants inhibit mating of the wild-type cells; the defect appears to be intrinsic to the cell for mutations in each of the genes. Apparently, none of the mutants is defective exclusively in division arrest by alpha-factor, as the sterility of none is suppressed by a temperature-sensitive cdc 28 mutation (the latter imposes division arrest at the correct cell cycle stage for mating). The mutants were examined for features that are inducible in MATa cells by alpha-factor (agglutinin synthesis as well as division arrest) and for the characteristics that constitutively distinguish MATa from MAT alpha cells (a-factor production, alpha-factor destruction). ste2 Mutants are defective specifically in the two inducible properties, whereas ste4, 5, 7, 8, 9, 11, and 12 mutants are defective, to varying degrees, in constitutive as well as inducible aspects. Mutations in ste8 and 9 assume a polar budding pattern unlike either MATa or MAT alpha cells but characteristic of MATa/alpha cells. This study defines seven genes that function in two cell types (MATa and alpha) to control the differentiation of cell type and one gene, ste2, that functions exclusively in MATa cells to mediate responsiveness to polypeptide hormone.     

CHS5, a gene involved in chitin synthesis and mating in Saccharomyces cerevisiae.

The CHS5 locus of Saccharomyces cerevisiae is important for wild-type levels of chitin synthase III activity. chs5 cells have reduced levels of this activity. To further understand the role of CHS5 in yeast, the CHS5 gene was cloned by complementation of the Calcofluor resistance phenotype of a chs5 mutant. Transformation of the mutant with a plasmid carrying CHS5 restored Calcofluor sensitivity, wild-type cell wall chitin levels, and chitin synthase III activity levels. DNA sequence analysis reveals that CHS5 encodes a unique polypeptide of 671 amino acids with a molecular mass of 73,642 Da. The predicted sequence shows a heptapeptide repeated 10 times, a carboxy-terminal lysine-rich tail, and some similarity to neurofilament proteins. The effects of deletion of CHS5 indicate that it is not essential for yeast cell growth; however, it is important for mating. Deletion of CHS3, the presumptive structural gene for chitin synthase III activity, results in a modest decrease in mating efficiency, whereas chs5delta cells exhibit a much stronger mating defect. However, chs5 cells produce more chitin than chs3 mutants, indicating that CHS5 plays a role in other processes besides chitin synthesis. Analysis of mating mixtures of chs5 cells reveals that cells agglutinate and make contact but fail to undergo cell fusion. The chs5 mating defect can be partially rescued by FUS1 and/or FUS2, two genes which have been implicated previously in cell fusion, but not by FUS3. In addition, mating efficiency is much lower in fus1 fus2 x chs5 than in fus1 fus2 x wild type crosses. Our results indicate that Chs5p plays an important role in the cell fusion step of mating.     

FUS1 regulates the opening and expansion of fusion pores between mating yeast

Mating yeast cells provide a genetically accessible system for the study of cell fusion. The dynamics of fusion pores between yeast cells were analyzed by following the exchange of fluorescent markers between fusion partners. Upon plasma membrane fusion, cytoplasmic GFP and DsRed diffuse between cells at rates proportional to the size of the fusion pore. GFP permeance measurements reveal that a typical fusion pore opens with a burst and then gradually expands. In some mating pairs, a sudden increase in GFP permeance was found, consistent with the opening of a second pore. In contrast, other fusion pores closed after permitting a limited amount of cytoplasmic exchange. Deletion of FUS1 from both mating partners caused a >10-fold reduction in the initial permeance and expansion rate of the fusion pore. Although fus1 mating pairs also have a defect in degrading the cell wall that separates mating partners before plasma membrane fusion, other cell fusion mutants with cell wall remodeling defects had more modest effects on fusion pore permeance. Karyogamy is delayed by >1 h in fus1 mating pairs, possibly as a consequence of retarded fusion pore expansion.     

The a-factor pheromone of Saccharomyces cerevisiae is essential for mating.

The Saccharomyces cerevisiae pheromone a-factor is produced by a cells and interacts with alpha cells to cause cell cycle arrest and other physiological responses associated with mating. Two a-factor structural genes, MFA1 and MFA2, have been previously cloned with synthetic probes based on the a-factor amino acid sequence (A. Brake, C. Brenner, R. Najarian, P. Laybourn, and J. Merryweather, cited in M.-J. Gething [ed.], Protein transport and secretion, 1985). We have examined the function of these genes in a-factor production and mating by construction and analysis of chromosomal null mutations. mfa1 and mfa2 single mutants each exhibited approximately half the wild-type level of a-factor activity and were proficient in mating, whereas the mfa1 mfa2 double mutant produced no a-factor and was unable to mate. These results demonstrate that both genes are functional, that each gene makes an equivalent contribution to the a-factor activity and mating capacity of a cells, and that a-factor plays an essential role in mating. Strikingly, exogenous a-factor did not alleviate the mating defect of the double mutant, suggesting that an a cell must be producing a-factor to be an effective mating partner.     

Fus2 localizes near the site of cell fusion and is required for both cell fusion and nuclear alignment during zygote formation

Zygote formation occurs through tightly coordinated cell and nuclear fusion events. Genetic evidence suggests that the FUS2 gene product promotes cell fusion during zygote formation in Saccharomyces cerevisiae, functioning with the Fus1 plasma membrane protein at or before cell wall and plasma membrane fusion. Here we report the sequence of the FUS2 gene, localization of Fus2 protein, and show that fus1 and fus2 mutants have distinct defects in cell fusion. FUS2 encodes a unique open reading frame of 617 residues that only is expressed in haploid cells in response to mating pheromone. Consistent with a role in cell fusion, Fus2 protein localizes with discrete structures that could be of cytoskeletal or vesicular origin that accumulate at the tip of pheromone-induced shmoos and at the junction of paired cells in zygotes. Fus2 is predicted to be a coiled-coil protein and fractionates with a 100,000 g pellet, suggesting that it is associated with cytoskeleton, membranes, or other macromolecular structures. Fus2 may interact with structures involved in the alignment of the nuclei during cell fusion, because fus2 mutants have strong defects in karyogamy and fail to orient microtubules between parental nuclei in zygotes. In contrast, fus1 mutants show no karyogamy defects. These, and other results suggest that Fus2 defines a novel cell fusion function and subcellular structure that is also required for the alignment of parental nuclei before nuclear fusion.     

Differential transmission of G1 cell cycle arrest and mating signals by Saccharomyces cerevisiae Ste5 mutants in the pheromone pathway.

Saccharomyces cerevisiae Ste5 is a scaffold protein that recruits many pheromone signaling molecules to sequester the pheromone pathway from other homologous mitogen-activated protein kinase pathways. G1 cell cycle arrest and mating are two different physiological consequences of pheromone signal transduction and Ste5 is required for both processes. However, the roles of Ste5 in G1 arrest and mating are not fully understood. To understand the roles of Ste5 better, we isolated 150 G1 cell cycle arrest defective STE5 mutants by chemical mutagenesis of the gene. Here, we found that two G1 cell cycle arrest defective STE5 mutants (ste5M(D248V) and ste5(delta-776)) retained mating capacity. When overproduced in a wild-type strain, several ste5 mutants also showed different dominant phenotypes for G1 arrest and mating. Isolation and characterization of the mutants suggested separable roles of Ste5 in G1 arrest and mating of S. cerevisiae. In addition, the roles of Asp-248 and Tyr-421, which are important for pheromone signal transduction were further characterized by site-directed mutagenesis studies.     

Two genes required for cell fusion during yeast conjugation: evidence for a pheromone-induced surface protein.

We characterized two genes, FUS1 and FUS2, which are required for fusion of Saccharomyces cerevisiae cells during conjugation. Mutations in these genes lead to an interruption of the mating process at a point just before cytoplasmic fusion; the partition dividing the mating pair remains undissolved several hours after the cells have initially formed a stable "prezygote." Fusion is only moderately impaired when the two parents together harbor one or two mutant fus genes, and it is severely compromised only when three or all four fus genes are inactivated. Cloning of FUS1 and FUS2 revealed that they share some functional homology; FUS1 on a high-copy number plasmid can partially suppress a fus2 mutant, and vice versa. FUS1 remains essentially unexpressed in vegetative cells, but is strongly induced by incubation of haploid cells with the appropriate mating pheromone. Immunofluorescence microscopy of alpha factor-induced a cells harboring a fus1-LACZ fusion showed the fusion protein to be localized at the cell surface, concentrated at one end of the cell (the shmoo tip). FUS1 maps near HIS4, and the intervening region (including BIK1, a gene required for nuclear fusion) was sequenced along with FUS1. The sequence of FUS1 revealed the presence of three copies of a hexamer (TGAAAC) conserved in the 5' noncoding regions of other pheromone-inducible genes. The deduced FUS1 protein sequence exhibits a striking concentration of serines and threonines at the amino terminus (46%; 33 of 71), followed by a 25-amino acid hydrophobic stretch and a predominantly hydrophilic carboxy terminus, which contains several potential N-glycosylation sites (Asn-X-Ser/Thr). This sequence suggests that FUS1 encodes a membrane-anchored glycoprotein with both N- and O-linked sugars.     

Rvs161p Interacts with Fus2p to Promote Cell Fusion in Saccharomyces cerevisiae

FUS7 was previously identified by a mutation that causes a defect in cell fusion in a screen for bilateral mating defects. Here we show that FUS7 is allelic to RVS161/END6, a gene implicated in a variety of processes including viability after starvation, endocytosis, and actin cytoskeletal organization. Two lines of evidence indicate that RVS161/END6's endocytic function is not required for cell fusion. First, several other endocytic mutants showed no cell fusion defects. Second, we isolated five function-specific alleles of RVS161/FUS7 that were defective for endocytosis, but not mating, and three alleles that were defective for cell fusion but not endocytosis. The organization of the actin cytoskeleton was normal in the cell fusion mutants, indicating that Rvs161p's function in cell fusion is independent of actin organization. The three to fourfold induction of RVS161 by mating pheromone and the localization of Rvs161p-GFP to the cell fusion zone suggested that Rvs161p plays a direct role in cell fusion. The phenotypes of double mutants, the coprecipitation of Rvs161p and Fus2p, and the fact that the stability of Fus2p was strongly dependent on Rvs161p's mating function lead to the conclusion that Rvs161p is required to interact with Fus2p for efficient cell fusion.     

Significance of C-terminal cysteine modifications to the biological activity of the Saccharomyces cerevisiae a-factor mating pheromone.

We have undertaken total synthesis of the Saccharomyces cerevisiae a-factor (NH2-YIIKGVFWDPAC[S-farnesyl]-COOCH3) and several Cys-12 analogs to determine the significance of S-farnesylation and carboxy-terminal methyl esterification to the biological activity of this lipopeptide mating pheromone. Replacement of either the farnesyl group or the carboxy-terminal methyl ester by a hydrogen atom resulted in marked reduction but not total loss of bioactivity as measured by a variety of assays. Moreover, both the farnesyl and methyl ester groups could be replaced by other substituents to produce biologically active analogs. The bioactivity of a-factor decreased as the number of prenyl units on the cysteine sulfur decreased from three to one, and an a-factor analog having the S-farnesyl group replaced by an S-hexadecanyl group was more active than an S-methyl a-factor analog. Thus, with two types of modifications, a-factor activity increased as the S-alkyl group became bulkier and more hydrophobic. MATa cells having deletions of the a-factor structural genes (mfal1 mfa2 mutants) were capable of mating with either sst2 or wild-type MAT alpha cells in the presence of exogenous a-factor, indicating that it is not absolutely essential for MATa cells to actively produce a-factor in order to mate. Various a-factor analogs were found to partially restore mating to these strains as well, and their relative activities in the mating restoration assay were similar to their activities in the other assays used in this study. Mating was not restored by addition of exogenous a-factor to a cross of a wild-type MAT alpha strain and a MATaste6 mutant, indicating a role of the STE6 gene product in mating in addition to its secretion of a-factor.     

A Role for a Protease in Morphogenic Responses during Yeast Cell Fusion

Cell fusion during yeast mating provides a model for signaling-controlled changes at the cell surface. We identified the AXL1 gene in a screen for genes required for cell fusion in both mating types during mating. AXL1 is a pheromone-inducible gene required for axial bud site selection in haploid yeast and for proteolytic maturation of a-factor. Two other bud site selection genes, RSR1, encoding a small GTPase, and BUD3, were also required for efficient cell fusion. Based on double mutant analysis, AXL1 in a MATα strain acted genetically in the same pathway with FUS2, a fusion-dedicated gene. Electron microscopy of axl1, rsr1, and fus2 prezygotes revealed similar defects in nuclear migration, vesicle accumulation, cell wall degradation, and membrane fusion during cell fusion. The axl1 and rsr1 mutants exhibited defects in pheromone-induced morphogenesis. AXL1 protease function was required in MATα strains for fusion during mating. The ability of the Rsr1p GTPase to cycle was required for efficient cell fusion, as it is for bud site selection. During conjugation, vegetative functions may be redeployed under the control of pheromone signaling for mating purposes. Since Rsr1p has been reported to physically associate with Cdc24p and Bem1p components of the pheromone response pathway, we suggest that the bud site selection genes Rsr1p and Axl1p may act to mediate pheromone control of Fus2p-based fusion events during mating.     

Lrg1p Is a Rho1 GTPase-activating protein required for efficient cell fusion in yeast

To identify additional cell fusion genes in Saccharomyces cerevisiae, we performed a high-copy suppressor screen of fus2Delta. Higher dosage of three genes, BEM1, LRG1, and FUS1, partially suppressed the fus2Delta cell fusion defect. BEM1 and FUS1 were high-copy suppressors of many cell-fusion-defective mutations, whereas LRG1 suppressed only fus2Delta and rvs161Delta. Lrg1p contains a Rho-GAP homologous region. Complete deletion of LRG1, as well as deletion of the Rho-GAP coding region, caused decreased rates of cell fusion and diploid formation comparable to that of fus2Delta. Furthermore, lrg1Delta caused a more severe mating defect in combination with other cell fusion mutations. Consistent with an involvement in cell fusion, Lrg1p localized to the tip of the mating projection. Lrg1p-GAP domain strongly and specifically stimulated the GTPase activity of Rho1p, a regulator of beta(1-3)-glucan synthase in vitro. beta(1-3)-glucan deposition was increased in lrg1Delta strains and mislocalized to the tip of the mating projection in fus2Delta strains. High-copy LRG1 suppressed the mislocalization of beta(1-3) glucan in fus2Delta strains. We conclude that Lrg1p is a Rho1p-GAP involved in cell fusion and speculate that it acts to locally inhibit cell wall synthesis to aid in the close apposition of the plasma membranes of mating cells.     

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.     

The Golgi-resident protease Kex2 acts in conjunction with Prm1 to facilitate cell fusion during yeast mating

The molecular machines that mediate cell fusion are unknown. Previously, we identified a multispanning transmembrane protein, Prm1 (pheromone-regulated membrane protein 1), that acts during yeast mating (Heiman, M.G., and P. Walter. 2000. J. Cell Biol. 151:719-730). Without Prm1, a substantial fraction of mating pairs arrest with their plasma membranes tightly apposed yet unfused. In this study, we show that lack of the Golgi-resident protease Kex2 strongly enhances the cell fusion defect of Prm1-deficient mating pairs and causes a mild fusion defect in otherwise wild-type mating pairs. Lack of the Kex1 protease but not the Ste13 protease results in similar defects. Deltakex2 and Deltakex1 fusion defects were suppressed by osmotic support, a trait shared with mutants defective in cell wall remodeling. In contrast, other cell wall mutants do not enhance the Deltaprm1 fusion defect. Electron microscopy of Deltakex2-derived mating pairs revealed novel extracellular blebs at presumptive sites of fusion. Kex2 and Kex1 may promote cell fusion by proteolytically processing substrates that act in parallel to Prm1 as an alternative fusion machine, as cell wall components, or both.