Characterization of the yeast amphiphysins Rvs161p and Rvs167p reveals roles for the Rvs heterodimer in vivo

We have used comprehensive synthetic lethal screens and biochemical assays to examine the biological role of the yeast amphiphysin homologues Rvs161p and Rvs167p, two proteins that play a role in regulation of the actin cytoskeleton, endocytosis, and sporulation. We found that unlike some forms of amphiphysin, Rvs161p-Rvs167p acts as an obligate heterodimer during vegetative growth and neither Rvs161p nor Rvs167p forms a homodimer in vivo. RVS161 and RVS167 have an identical set of 49 synthetic lethal interactions, revealing functions for the Rvs proteins in cell polarity, cell wall synthesis, and vesicle trafficking as well as a shared role in mating. Consistent with these roles, we show that the Rvs167p-Rvs161p heterodimer, like its amphiphysin homologues, can bind to phospholipid membranes in vitro, suggesting a role in vesicle formation and/or fusion. Our genetic screens also reveal that the interaction between Abp1p and the Rvs167p Src homology 3 (SH3) domain may be important under certain conditions, providing the first genetic evidence for a role for the SH3 domain of Rvs167p. Our studies implicate heterodimerization of amphiphysin family proteins in various functions related to cell polarity, cell integrity, and vesicle trafficking during vegetative growth and the mating response.     

end5, end6, and end7: mutations that cause actin delocalization and block the internalization step of endocytosis in Saccharomyces cerevisiae.

Four mutants defective in endocytosis were isolated by screening a collection of temperature-sensitive yeast mutants. Three mutations define new END genes: end5-1, end6-1, and end7-1. The fourth mutation is in END4, a gene identified previously. The end5-1, end6-1, and end7-1 mutations do not affect vacuolar protein localization, indicating that the defect in each mutant is specific for internalization at the plasma membrane. Interestingly, localization of actin patches on the plasma membrane is affected in each of the mutants. end5-1, end6-1, and end7-1 are allelic to VRP1, RVS161, and ACT1, respectively. VRP1 and RVS161 are required for correct actin localization and ACT1 encodes actin. To our surprise, the end6-1 mutation fails to complement the act1-1 mutation. Disruption of the RVS167 gene, which is homologous to END6/RVS161 and which is also required for correct actin localization, also blocks endocytosis. The end7-1 mutant allele has a glycine 48 to aspartic acid substitution in the DNase I-binding loop of actin. We propose that Vrp1p, Rvs161p, and Rvs167p are components of a cytoskeletal structure that contains actin and fimbrin and that is required for formation of endocytic vesicles at the plasma membrane.     

Dynamic localization of yeast Fus2p to an expanding ring at the cell fusion junction during mating

Fus2p is a pheromone-induced protein associated with the amphiphysin homologue Rvs161p, which is required for cell fusion during mating in Saccharomyces cerevisiae. We constructed a functional Fus2p-green fluorescent protein (GFP), which exhibits highly dynamic localization patterns in pheromone-responding cells (shmoos): diffuse nuclear, mobile cytoplasmic dots and stable cortical patches concentrated at the shmoo tip. In mitotic cells, Fus2p-GFP is nuclear but becomes cytoplasmic as cells form shmoos, dependent on the Fus3p protein kinase and high levels of pheromone signaling. The rapid cytoplasmic movement of Fus2p-GFP dots requires Rvs161p and polymerized actin and is aberrant in mutants with compromised actin organization, which suggests that the Fus2p dots are transported along actin cables, possibly in association with vesicles. Maintenance of Fus2p-GFP patches at the shmoo tip cortex is jointly dependent on actin and a membrane protein, Fus1p, which suggests that Fus1p is an anchor for Fus2p. In zygotes, Fus2p-GFP forms a dilating ring at the cell junction, returning to the nucleus at the completion of cell fusion.     

End4p/Sla2p Interacts with Actin-associated Proteins for Endocytosis in Saccharomyces cerevisiae

end4–1 was isolated as a temperature-sensitive endocytosis mutant. We cloned and sequenced END4 and found that it is identical to SLA2/MOP2. This gene is required for growth at high temperature, viability in the absence of Abp1p, polarization of the cortical actin cytoskeleton, and endocytosis. We used a mutational analysis of END4 to correlate in vivo functions with regions of End4p and we found that two regions of End4p participate in endocytosis but that the talin-like domain of End4p is dispensable. The N-terminal domain of End4p is required for growth at high temperature, endocytosis, and actin organization. A central coiled-coil domain of End4p is necessary for formation of a soluble sedimentable complex. Furthermore, this domain has an endocytic function that is redundant with the function(s) of ABP1 and SRV2. The endocytic function of Abp1p depends on its SH3 domain. In addition we have isolated a recessive negative allele of SRV2 that is defective for endocytosis. Combined biochemical, functional, and genetic analysis lead us to propose that End4p may mediate endocytosis through interaction with other actin-associated proteins, perhaps Rvs167p, a protein essential for endocytosis.     

Distinct Morphological Phenotypes of Cell Fusion Mutants

Cell fusion in yeast is the process by which two haploid cells fuse to form a diploid zygote. To dissect the pathway of cell fusion, we phenotypically and genetically characterized four cell fusion mutants, fus6/spa2, fus7/rvs161, fus1, and fus2. First, we examined the complete array of single and double mutants. In all cases but one, double mutants exhibited stronger cell fusion defects than single mutants. The exception was rvs161Δ fus2Δ, suggesting that Rvs161p and Fus2p act in concert. Dosage suppression analysis showed that Fus1p and Fus2p act downstream or parallel to Rvs161p and Spa2p. Second, electron microscopic analysis was used to define the mutant defects in cell fusion. In wild-type prezygotes vesicles were aligned and clustered across the cell fusion zone. The vesicles were associated with regions of cell wall thinning. Analysis of Fus⁻ zygotes indicated that Fus1p was required for the normal localization of the vesicles to the zone of cell fusion, and Spa2p facilitated their clustering. In contrast, Fus2p and Rvs161p appeared to act after vesicle positioning. These findings lead us to propose that cell fusion is mediated in part by the localized release of vesicles containing components essential for cell fusion.     

Rvs161p and Rvs167p, the two yeast amphiphysin homologs, function together in vivo

Mutations in RVS161 and RVS167, the two yeast amphiphysin homologs, cause very similar growth phenotypes, a depolarized actin cytoskeleton, and a defect in the internalization step of endocytosis. Rvs161p and Rvs167p have been shown to interact in the two-hybrid system, but their localization in the cell may be different thus raising the question whether the interaction is physiologically relevant. Here we demonstrate that the two proteins function together in vivo. We find that the steady state level of Rvs167p is strongly reduced in an rvs161Delta strain. Similarly, the level of Rvs161p is strongly reduced in an rvs167Delta strain. We demonstrate that these reduced protein levels at steady state are due to a decreased stability of either Rvs protein in the absence of the other protein. Furthermore, we find that the amount and ratio of Rvs161p and Rvs167p are critical parameters for receptor-mediated endocytosis. In addition, by using the two-hybrid system we show that the interaction of Rvs167p with actin is not abolished in an abp1Delta strain suggesting that Abp1p is not essential for this interaction.     

Characterizing the sphingolipid signaling pathway that remediates defects associated with loss of the yeast amphiphysin-like orthologs, Rvs161p and Rvs167p

Loss of function of either the RVS161 or RVS167 Saccharomyces cerevisiae amphiphysin-like gene confers similar growth phenotypes that can be suppressed by mutations in sphingolipid biosynthesis. We performed a yeast two-hybrid screen using Rvs161p as bait to uncover proteins involved in this sphingolipid-dependent suppressor pathway. In the process, we have demonstrated a direct physical interaction between Rvs167p and the two-hybrid interacting proteins, Acf2p, Gdh3p, and Ybr108wp, while also elucidating the Rvs167p amino acid domains to which these proteins bind. By using subcellular fractionation, we demonstrate that Rvs167p, Ybr108wp, Gdh3p, and Acf2p all localize to Rvs161p-containing lipid rafts, thus placing them within a single compartment that should facilitate their interactions. Moreover, our results suggest that Acf2p and Gdh3p functions are needed for suppressor pathway activity. To determine pathway mechanisms further, we examined the localization of Rvs167p in suppressor mutants. These studies reveal roles for Rvs161p and the very long chain fatty acid elongase, Sur4p, in the localization and/or stability of Rvs167p. Previous yeast studies showed that rvs defects could be suppressed by changes in sphingolipid metabolism brought about by deleting SUR4 (Desfarges, L., Durrens, P., Juguelin, H., Cassagne, C., Bonneu, M., and Aigle, M. (1993) Yeast 9, 267-277). Using rvs167 sur4 and rvs161 sur4 double null cells as models to study suppressor pathway activity, we demonstrate that loss of SUR4 does not remediate the steady-state actin cytoskeletal defects of rvs167 or rvs161 cells. Moreover, suppressor activity does not require the function of the actin-binding protein, Abp1p, or Sla1p, a protein that is thought to regulate assembly of the cortical actin cytoskeleton. Based on our results, we suggest that sphingolipid-dependent suppression of rvs defects may not work entirely through regulating changes in actin organization.     

Rvs161p and sphingolipids are required for actin repolarization following salt stress

In Saccharomyces cerevisiae, the actin cytoskeleton is depolarized by NaCl stress. In this study, the response was maximal after 30 min, and then actin patches repolarized. Rvs161p was required for actin repolarization because the rvs161Δ mutant did not repolarize actin patches after growth in a salt medium. Mutations suppressing the rvs161Δ-related salt sensitivity all occurred in genes required for sphingolipid biosynthesis: FEN1, SUR4, SUR2, SUR1, and IPT1. These suppressors also suppressed act1-1-related salt sensitivity and the defect in actin repolarization of the rvs161Δ mutant, providing a link between sphingolipids and actin polarization. Indeed, deletion of the suppressor genes suppressed the rvs161Δ defect in actin repolarization in two ways: either actin was not depolarized at the wild-type level in a set of suppressor mutants, or actin was repolarized in the absence of Rvs161p in the other suppressor mutants. Rvs161p was localized as cortical patches that concentrated at polarization sites, i.e., bud emergence and septa, and was found to be associated with lipid rafts. An important link between sphingolipids and actin polarization is that Rvs161p was required for actin repolarization and was found to be located in lipid rafts.     

Human BIN3 complements the F-actin localization defects caused by loss of Hob3p, the fission yeast homolog of Rvs161p

The BAR adaptor proteins encoded by the RVS167 and RVS161 genes from Saccharomyces cerevisiae form a complex that regulates actin, endocytosis, and viability following starvation or osmotic stress. In this study, we identified a human homolog of RVS161, termed BIN3 (bridging integrator-3), and a Schizosaccharomyces pombe homolog of RVS161, termed hob3+ (homolog of Bin3). In human tissues, the BIN3 gene was expressed ubiquitously except for brain. S. pombe cells lacking Hob3p were often multinucleate and characterized by increased amounts of calcofluor-stained material and mislocalized F-actin. For example, while wild-type cells localized F-actin to cell ends during interphase, hob3Delta mutants had F-actin patches distributed randomly around the cell. In addition, medial F-actin rings were rarely found in hob3Delta mutants. Notably, in contrast to S. cerevisiae rvs161Delta mutants, hob3Delta mutants showed no measurable defects in endocytosis or response to osmotic stress, yet hob3+ complemented the osmosensitivity of a rvs161Delta mutant. BIN3 failed to rescue the osmosensitivity of rvs161Delta, but the actin localization defects of hob3Delta mutants were completely rescued by BIN3 and partially rescued by RVS161. These findings suggest that hob3+ and BIN3 regulate F-actin localization, like RVS161, but that other roles for this gene have diverged somewhat during evolution.     

Pheromone-induced polarization is dependent on the Fus3p MAPK acting through the formin Bni1p

During mating, budding yeast cells reorient growth toward the highest concentration of pheromone. Bni1p, a formin homologue, is required for this polarized growth by facilitating cortical actin cable assembly. Fus3p, a pheromone-activated MAP kinase, is required for pheromone signaling and cell fusion. We show that Fus3p phosphorylates Bni1p in vitro, and phosphorylation of Bni1p in vivo during the pheromone response is dependent on Fus3p. fus3 mutants exhibited multiple phenotypes similar to bni1 mutants, including defects in actin and cell polarization, as well as Kar9p and cytoplasmic microtubule localization. Disruption of the interaction between Fus3p and the receptor-associated Galpha subunit caused similar mutant phenotypes. After pheromone treatment, Bni1p-GFP and Spa2p failed to localize to the cortex of fus3 mutants, and cell wall growth became completely unpolarized. Bni1p overexpression suppressed the actin assembly, cell polarization, and cell fusion defects. These data suggest a model wherein activated Fus3p is recruited back to the cortex, where it activates Bni1p to promote polarization and cell fusion.     

Regulation of the yeast amphiphysin homologue Rvs167p by phosphorylation

The yeast amphiphysin homologue Rvs167p plays a role in regulation of the actin cytoskeleton, endocytosis, and sporulation. Rvs167p is a phosphoprotein in vegetatively growing cells and shows increased phosphorylation upon treatment with mating pheromone. Previous work has shown that Rvs167p can be phosphorylated in vitro by the cyclin-dependent kinase Pho85p complexed with its cyclin Pcl2p. Using chymotryptic phosphopeptide mapping, we have identified the sites on which Rvs167p is phosphorylated in vitro by Pcl2p-Pho85p. We have shown that these same sites are phosphorylated in vivo during vegetative growth and that phosphorylation at two of these sites is Pcl-Pho85p dependent. In cells treated with mating pheromone, the MAP kinase Fus3p is needed for full phosphorylation of Rvs167p. Functional genomics and genetics experiments revealed that mutation of other actin cytoskeleton genes compromises growth of a strain in which phosphorylation of Rvs167p is blocked by mutation. Phosphorylation of Rvs167p inhibits its interaction in vitro with Las17p, an activator of the Arp2/3 complex, as well as with a novel protein, Ymr192p. Our results suggest that phosphorylation of Rvs167p by a cyclin-dependent kinase and by a MAP kinase is an important mechanism for regulating protein complexes involved in actin cytoskeleton function.     

Identification and Characterization of Rvs162/Rvs167-3, a Novel N-BAR Heterodimer in the Human Fungal Pathogen Candida albicans

Membrane reshaping resides at the core of many important cellular processes, and among its mediators are the BAR (Bin, Amphiphysin, Rvs) domain-containing proteins. We have explored the diversity and function of the Rvs BAR proteins in Candida albicans and identified a novel family member, Rvs167-3 (orf19.1861). We show that Rvs167-3 specifically interacts with Rvs162 to form a stable BAR heterodimer able to bind liposomes in vitro. A second, distinct heterodimer is formed by the canonical BAR proteins Rvs161 and Rvs167. Purified Rvs161/Rvs167 complex also binds liposomes, indicating that C. albicans expresses two functional BAR heterodimers. We used live-cell imaging to localize green fluorescent protein (GFP)-tagged Rvs167-3 and Rvs167 and show that both proteins concentrate in small cortical spots. However, while Rvs167 strictly colocalizes with the endocytic marker protein Abp1, we do not observe any colocalization of Rvs167-3 with sites of endocytosis marked by Abp1. Furthermore, the rvs167-3Δ/Δ mutant is not defective in endocytosis and strains lacking Rvs167-3 or its partner Rvs162 do not display increased sensitivity to high salt concentrations or decreased cell wall integrity, phenotypes which have been observed for rvs167Δ/Δ and rvs161Δ/Δ strains and which are linked to endocytosis defects. Taken together, our results indicate different roles for the two BAR heterodimers in C. albicans: the canonical Rvs161/Rvs167 heterodimer functions in endocytosis, whereas the novel Rvs162/Rvs167-3 heterodimer seems not to be involved in this process. Nevertheless, despite their different roles, our phenotypic analysis revealed a genetic interaction between the two BAR heterodimers, suggesting that they may have related but distinct membrane-associated functions.     

Cortical recruitment and nuclear-cytoplasmic shuttling of Scd5p, a protein phosphatase-1-targeting protein involved in actin organization and endocytosis

Scd5p regulates endocytosis and cortical actin organization as a targeting subunit for the Ser/Thr protein phosphatase-1 (PP1) in yeast. To identify localization signals in Scd5p required for cell surface recruitment, visualization of GFP-tagged Scd5 truncations and deletions was performed. Scd5p contains a PP1 binding site, a 3-repeat region of 20 amino acids (3R), and a 9-repeat region of 12 amino acids (9R). We found that the 9R is critical for cortical localization of Scd5p, but cortical recruitment is not essential for Scd5p's function in actin organization and endocytosis. We propose that Scd5p can target PP1 to endocytic factors in the cytoplasm that have been disassembled and/or inactivated by phosphorylation. We also found that Scd5p undergoes nuclear-cytoplasmic shuttling in a Crm1p-dependent manner. Scd5p-DeltaCT lacking the 9R region and its nuclear export signal (NES) accumulates in the nucleus, causing cortical actin and endocytic defects. Cytoplasmic localization and function of Scd5p-DeltaCT is restored by NES addition. However, removal of Scd5p's nuclear localization signal prevents nuclear entry, but endocytosis and actin organization remain relatively normal. These results indicate that nuclear-cytoplasmic shuttling is not required for regulation of Scd5p's cortical function and suggest that Scd5p has an independent nuclear function.     

Specific protein targeting during cell differentiation: polarized localization of Fus1p during mating depends on Chs5p in Saccharomyces cerevisiae

In budding yeast, chs5 mutants are defective in chitin synthesis and cell fusion during mating. Chs5p is a late-Golgi protein required for the polarized transport of the chitin synthase Chs3p to the membrane. Here we show that Chs5p is also essential for the polarized targeting of Fus1p, but not of other cell fusion proteins, to the membrane during mating.     

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.     

Dissecting BAR Domain Function in the Yeast Amphiphysins Rvs161 and Rvs167 during Endocytosis

BAR domains are protein modules that bind to membranes and promote membrane curvature. One type of BAR domain, the N-BAR domain, contains an additional N-terminal amphipathic helix, which contributes to membrane-binding and bending activities. The only known N-BAR-domain proteins in the budding yeast Saccharomyces cerevisiae, Rvs161 and Rvs167, are required for endocytosis. We have explored the mechanism of N-BAR-domain function in the endocytosis process using a combined biochemical and genetic approach. We show that the purified Rvs161–Rvs167 complex binds to liposomes in a curvature-independent manner and promotes tubule formation in vitro. Consistent with the known role of BAR domain polymerization in membrane bending, we found that Rvs167 BAR domains interact with each other at cortical actin patches in vivo. To characterize N-BAR-domain function in endocytosis, we constructed yeast strains harboring changes in conserved residues in the Rvs161 and Rvs167 N-BAR domains. In vivo analysis of the rvs endocytosis mutants suggests that Rvs proteins are initially recruited to sites of endocytosis through their membrane-binding ability. We show that inappropriate regulation of complex sphingolipid and phosphoinositide levels in the membrane can impinge on Rvs function, highlighting the relationship between membrane components and N-BAR-domain proteins in vivo.     

In vivo analysis of the domains of yeast Rvs167p suggests Rvs167p function is mediated through multiple protein interactions.

Morphological changes during cell division in the yeast Saccharomyces cerevisiae are controlled by cell-cycle regulators. The Pcl-Pho85p kinase complex has been implicated in the regulation of the actin cytoskeleton at least in part through Rvs167p. Rvs167p consists of three domains called BAR, GPA, and SH3. Using a two-hybrid assay, we demonstrated that each region of Rvs167p participates in protein-protein interactions: the BAR domain bound the BAR domain of another Rvs167p protein and that of Rvs161p, the GPA region bound Pcl2p, and the SH3 domain bound Abp1p. We identified Rvs167p as a Las17p/Bee1p-interacting protein in a two-hybrid screen and showed that Las17p/Bee1p bound the SH3 domain of Rvs167p. We tested the extent to which the Rvs167p protein domains rescued phenotypes associated with deletion of RVS167: salt sensitivity, random budding, and endocytosis and sporulation defects. The BAR domain was sufficient for full or partial rescue of all rvs167 mutant phenotypes tested but not required for the sporulation defect for which the SH3 domain was also sufficient. Overexpression of Rvs167p inhibits cell growth. The BAR domain was essential for this inhibition and the SH3 domain had only a minor effect. Rvs167p may link the cell cycle regulator Pcl-Pho85p kinase and the actin cytoskeleton. We propose that Rvs167p is activated by phosphorylation in its GPA region by the Pcl-Pho85p kinase. Upon activation, Rvs167p enters a multiprotein complex, making critical contacts in its BAR domain and redundant or minor contacts with its SH3 domain.     

The tetraspan protein Dni1p is required for correct membrane organization and cell wall remodelling during mating in Schizosaccharomyces pombe

In fungi, success of mating requires that both cells agglutinate, modify their extracellular envelopes, and fuse their plasma membranes and nuclei to produce a zygote. Here we studied the role of the Schizosaccharomyces pombe Dni1 protein in the cell fusion step of mating. Dni1p is a tetraspan protein bearing a conserved cystein motif similar to that present in fungal claudin-related proteins. Dni1p expression is induced during mating and Dni1p concentrates as discrete patches at the cell–cell contact area and along the mating bridge. Proper Dni1p localization depends on Fus1p, actin and integrity of lipid rafts. In dni1 Δ mutants, cell differentiation and agglutination are as efficient as in the wild-type strain, but cell fusion is significantly reduced at temperatures above 25°C. We found that the defect in cell fusion was not associated with an altered cytoskeleton, with an abnormal distribution of Fus1p, or with a defect in calcium accumulation, but with a severe disorganization of the plasma membrane and cell wall at the area of cell–cell contact. These results show that Dni1p plays a relevant role in co-ordinating membrane organization and cell wall remodelling during mating, a function that has not been described for other proteins in the fission yeast.     

The Class V Myosin Myo2p Is Required for Fus2p Transport and Actin Polarization during the Yeast Mating Response

Mating yeast cells remove their cell walls and fuse their plasma membranes in a spatially restricted cell contact region. Cell wall removal is dependent on Fus2p, an amphiphysin-associated Rho-GEF homolog. As mating cells polarize, Fus2p-GFP localizes to the tip of the mating projection, where cell fusion will occur, and to cytoplasmic puncta, which show rapid movement toward the tip. Movement requires polymerized actin, whereas tip localization is dependent on both actin and a membrane protein, Fus1p. Here, we show that Fus2p-GFP movement is specifically dependent on Myo2p, a type V myosin, and not on Myo4p, another type V myosin, or Myo3p and Myo5p, type I myosins. Fus2p-GFP tip localization and actin polarization in shmoos are also dependent on Myo2p. A temperature-sensitive tropomyosin mutation and Myo2p alleles that specifically disrupt vesicle binding caused rapid loss of actin patch organization, indicating that transport is required to maintain actin polarity. Mutant shmoos lost actin polarity more rapidly than mitotic cells, suggesting that the maintenance of cell polarity in shmoos is more sensitive to perturbation. The different velocities, differential sensitivity to mutation and lack of colocalization suggest that Fus2p and Sec4p, another Myo2p cargo associated with exocytotic vesicles, reside predominantly on different cellular organelles.     

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.