Prm1 Targeting to Contact Sites Enhances Fusion during Mating in Saccharomyces cerevisiae

Prm1 is a pheromone-regulated membrane glycoprotein involved in the plasma membrane fusion event of Saccharomyces cerevisiae mating. Although this function suggests that Prm1 should act at contact sites in pairs of mating yeast cells where plasma membrane fusion occurs, only a small percentage of the total Prm1 was actually detected on the plasma membrane. We therefore investigated the intracellular transport of Prm1 and how this transport contributes to cell fusion. Two Prm1 chimeras that were sorted away from the contact site had reduced fusion activity, indicating that Prm1 indeed functions at contact sites. However, most Prm1 is located in endosomes and other cytoplasmic organelles and is targeted to vacuoles for degradation. Mutations in a putative endocytosis signal in a cytoplasmic loop partially stabilized the Prm1 protein and caused it to accumulate on the plasma membrane, but this endocytosis mutant actually had reduced mating activity. When Prm1 was expressed from a galactose-regulated promoter and its synthesis was repressed at the start of mating, vanishingly small amounts of Prm1 protein remained at the time when the plasma membranes came into contact. Nevertheless, this stable pool of Prm1 was retained at polarized sites on the plasma membrane and was sufficient to promote plasma membrane fusion. Thus, the amount of Prm1 expressed in mating yeast is far in excess of the amount required to facilitate fusion.Membrane fusion has been studied extensively in the context of viral infection and intracellular membrane fusion. These fusion events are mediated by fusases—proteins that mediate membrane fusion. Some of the best-studied fusases are the SNAREs (soluble N-ethylmaleimide-sensitive factors) that mediate fusion of intracellular organelles and the hemagglutinin (HA) protein of influenza virus that mediates fusion of the viral envelope membrane with host endosomes (13). However, little is known about how the plasma membranes of two cells fuse during cell fusion.Cell fusion is essential for the development of multicellular organisms. Some cell fusion processes involve a single pair of cells, as in sperm-egg fusion. Many other developmental processes require multiple fusion events, as in fusion of myoblasts for muscle formation. However, all fusion events must overcome a common obstacle—maintaining the integrity and selective permeability of the two plasma membranes while fusing the hydrophobic cores of their phospholipid bilayers.We study cell fusion in mating pairs of the yeast Saccharomyces cerevisiae. This organism offers a genetically tractable model amenable to many biochemical and cell biological assays. The mating pathway in yeast is comprised of 5 steps: pheromone signaling, adhesion, degradation of the intervening cell walls, plasma membrane fusion, and karyogamy. S. cerevisiae has two haploid mating types: MATa and MATα. Haploid cells secrete pheromones that bind to G-protein-coupled receptors on the surface of cells of the opposite mating type. Pheromone binding activates a signaling cascade that causes cell cycle arrest, expression of pheromone-inducible genes, and polarized growth to form a mating projection (or shmoo tip). The binding of two cells of opposite mating type to form a mating pair is mediated by complementary agglutinins located on the shmoo tips. Then, the cell walls of the two cells are joined to form a unified wall protecting the mating pair, and the walls between the two cells are degraded. This allows the plasma membranes to come into contact and fuse. The initial fusion pore between cells expands to allow cytoplasmic mixing and, ultimately, karyogamy. After mating is complete, the mitotic cell cycle resumes, and a diploid daughter cell buds from the neck connecting the two parent cells (5, 30).This work focuses on Prm1, a glycoprotein that promotes the plasma membrane fusion step of mating. PRM1 was discovered in a bioinformatic screen designed to identify Prm (pheromone-regulated membrane) proteins (11). Prm1 has four transmembrane domains and functions as a disulfide-linked dimer (20). Prm1-deficient mating pairs experience one of three fates: arrest as late prezygotes (unfused mating pairs with no intervening cell walls), lysis once their plasma membranes come into contact, or fusion. Electron microscopy revealed that the two plasma membranes in a late prezygote were only ∼8 nm apart but did not fuse. Additional studies showed that ∼30% of prm1Δ mating pairs lyse after membrane contact (1, 14). However, 50% of prm1Δ mating pairs fuse on standard yeast extract-peptone-dextrose (YPD) medium, implying that Prm1 is important, but not required, for fusion. Mating becomes more dependent upon Prm1 activity if Ca²⁺ or ergosterol is limiting (1, 15).On the basis of its apparent role in membrane fusion, Prm1 should be targeted to the contact sites where membranes fuse. Surprisingly, only a small amount of Prm1 was found at contact sites, and even less was at shmoo tips or at bud tips in mitotic cells expressing Prm1 from a constitutive promoter. These observations prompted further investigation of Prm1''s intracellular transport. The results revealed that Prm1 does indeed function at contact sites. However, except for the small pool that promotes fusion, Prm1 proteins are transported to vacuoles and rapidly degraded.     

Vegetative hyphal fusion is not essential for plant infection by Fusarium oxysporum

Vegetative hyphal fusion (VHF) is a ubiquitous phenomenon in filamentous fungi whose biological role is poorly understood. In Neurospora crassa, the mitogen-activated protein kinase (MAPK) Mak-2 and the WW domain protein So are required for efficient VHF. A MAPK orthologous to Mak-2, Fmk1, was previously shown to be essential for root penetration and pathogenicity of the vascular wilt fungus Fusarium oxysporum. Here we took a genetic approach to test two hypotheses, that (i) VHF and plant infection have signaling mechanisms in common and (ii) VHF is required for efficient plant infection. F. oxysporum mutants lacking either Fmk1 or Fso1, an orthologue of N. crassa So, were impaired in the fusion of vegetative hyphae and microconidial germ tubes. Δfmk1 Δfso1 double mutants exhibited a more severe fusion phenotype than either single mutant, indicating that the two components function in distinct pathways. Both Δfso1 and Δfmk1 strains were impaired in the formation of hyphal networks on the root surface, a process associated with extensive VHF. The Δfso1 mutants exhibited slightly reduced virulence in tomato fruit infection assays but, in contrast to Δfmk1 strains, were still able to perform functions associated with invasive growth, such as secretion of pectinolytic enzymes or penetration of cellophane sheets, and to infect tomato plants. Thus, although VHF per se is not essential for plant infection, both processes have some signaling components in common, suggesting an evolutionary relationship between the underlying cellular mechanisms.     

Identification of Kel1p,a Kelch Domain-containing Protein Involved in Cell Fusion and Morphology in Saccharomyces cerevisiae

We showed previously that protein kinase C, which is required to maintain cell integrity, negatively regulates cell fusion (Philips, J., and I. Herskowitz. 1997. J. Cell Biol. 138:961–974). To identify additional genes involved in cell fusion, we looked for genes whose overexpression relieved the defect caused by activated alleles of Pkc1p. This strategy led to the identification of a novel gene, KEL1, which encodes a protein composed of two domains, one containing six kelch repeats, a motif initially described in the Drosophila protein Kelch (Xue, F., and L. Cooley. 1993. Cell. 72:681– 693), and another domain predicted to form coiled coils. Overexpression of KEL1 also suppressed the defect in cell fusion of spa2Δ and fps1Δ mutants. KEL2, which corresponds to ORF YGR238c, encodes a protein highly similar to Kel1p. Its overexpression also suppressed the mating defect associated with activated Pkc1p. Mutants lacking KEL1 exhibited a moderate defect in cell fusion that was exacerbated by activated alleles of Pkc1p or loss of FUS1, FUS2, or FPS1, but not by loss of SPA2. kel1Δ mutants form cells that are elongated and heterogeneous in shape, indicating that Kel1p is also required for proper morphology during vegetative growth. In contrast, kel2Δ mutants were not impaired in cell fusion or morphology. Both Kel1p and Kel2p localized to the site where cell fusion occurs during mating and to regions of polarized growth during vegetative growth. Coimmunoprecipitation and two-hybrid analyses indicated that Kel1p and Kel2p physically interact. We conclude that Kel1p has a role in cell morphogenesis and cell fusion and may antagonize the Pkc1p pathway.     

Overlapping Functions for Two G Protein α Subunits in Neurospora Crassa

Heterotrimeric G proteins, consisting of α, β and γ subunits, mediate a variety of signaling pathways in eukaryotes. We have previously identified two genes, gna-1 and gna-2, that encode G protein α subunits in the filamentous fungus Neurospora crassa. Mutation of gna-1 results in female infertility and sensitivity to hyperosmotic media. In this study, we investigate the expression and functions of gna-2. Results from Western analysis and measurements of gna-2 promoter-lacZ fusion activity indicate that gna-2 is expressed during the vegetative and sexual cycle of N. crassa in both A and a mating types. Activating mutations predicted to abolish the GTPase activity of GNA-2 cause subtle defects in aerial hyphae formation and conidial germination. Extensive phenotypic analysis of Δgna-2 strains did not reveal abnormalities during vegetative or sexual development. In contrast, deletion of gna-2 in a Δgna-1 strain accentuates the Δgna-1 phenotypes. Δgna-1 Δgna-2 strains have a slower rate of hyphal apical extension than Δgna-1 strains on hyperosmotic media. Moreover, Δgna-1 Δgna-2 mutants have more pronounced defects in female fertility than Δgna-1 strains. We propose that gna-1 and gna-2 have overlapping functions and may constitute a gene family. This is the first report of G protein α subunits with overlapping functions in eukaryotic microbes.     

Prm3p Is a Pheromone-induced Peripheral Nuclear Envelope Protein Required for Yeast Nuclear Fusion

Nuclear membrane fusion is the last step in the mating pathway of the yeast Saccharomyces cerevisiae. We adapted a bioinformatics approach to identify putative pheromone-induced membrane proteins potentially required for nuclear membrane fusion. One protein, Prm3p, was found to be required for nuclear membrane fusion; disruption of PRM3 caused a strong bilateral defect, in which nuclear congression was completed but fusion did not occur. Prm3p was localized to the nuclear envelope in pheromone-responding cells, with significant colocalization with the spindle pole body in zygotes. A previous report, using a truncated protein, claimed that Prm3p is localized to the inner nuclear envelope. Based on biochemistry, immunoelectron microscopy and live cell microscopy, we find that functional Prm3p is a peripheral membrane protein exposed on the cytoplasmic face of the outer nuclear envelope. In support of this, mutations in a putative nuclear localization sequence had no effect on full-length protein function or localization. In contrast, point mutations and deletions in the highly conserved hydrophobic carboxy-terminal domain disrupted both protein function and localization. Genetic analysis, colocalization, and biochemical experiments indicate that Prm3p interacts directly with Kar5p, suggesting that nuclear membrane fusion is mediated by a protein complex.     

Evidence for Transceptor Function of Cellodextrin Transporters in Neurospora crassa

Neurospora crassa colonizes burnt grasslands and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source to cellulose, N. crassa dramatically up-regulates expression and secretion of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Previously, we have shown that a N. crassa mutant carrying deletions of three β-glucosidase enzymes (Δ3βG) lacks β-glucosidase activity, but efficiently induces cellulase gene expression and cellulolytic activity in the presence of cellobiose as the sole carbon source. These observations indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression and activity in N. crassa. Here, we show that in N. crassa, two cellodextrin transporters, CDT-1 and CDT-2, contribute to cellulose sensing. A N. crassa mutant carrying deletions for both transporters is unable to induce cellulase gene expression in response to crystalline cellulose. Furthermore, a mutant lacking genes encoding both the β-glucosidase enzymes and cellodextrin transporters (Δ3βGΔ2T) does not induce cellulase gene expression in response to cellobiose. Point mutations that severely reduce cellobiose transport by either CDT-1 or CDT-2 when expressed individually do not greatly impact cellobiose induction of cellulase gene expression. These data suggest that the N. crassa cellodextrin transporters act as “transceptors” with dual functions - cellodextrin transport and receptor signaling that results in downstream activation of cellulolytic gene expression. Similar mechanisms of transceptor activity likely occur in related ascomycetes used for industrial cellulase production.     

Shared and independent roles for a Galpha(i) protein and adenylyl cyclase in regulating development and stress responses in Neurospora crassa

Growth and development are regulated using cyclic AMP (cAMP)-dependent and -independent pathways in Neurospora crassa. The cr-1 adenylyl cyclase mutant lacks detectable cAMP and exhibits numerous defects, including colonial growth habit, short aerial hyphae, premature conidiation on plates, inappropriate conidiation in submerged culture, and increased thermotolerance. Evidence suggests that the heterotrimeric Gα protein GNA-1 is a direct positive regulator of adenylyl cyclase. Δgna-1 strains are female-sterile, and Δgna-1 strains have reduced apical extension rates on normal and hyperosmotic medium, greater resistance to oxidative and heat stress, and stunted aerial hyphae compared to the wild-type strain. In this study, a Δgna-1 cr-1 double mutant was analyzed to differentiate cAMP-dependent and -independent signaling pathways regulated by GNA-1. Δgna-1 cr-1 mutants have severely restricted colonial growth and do not produce aerial hyphae on plates or in standing liquid cultures. Addition of cAMP to plates or standing liquid cultures rescues cr-1, but not Δgna-1 cr-1, defects, which is consistent with previous results demonstrating that Δgna-1 mutants do not respond to exogenous cAMP. The females of all strains carrying the Δgna-1 mutation are sterile; however, unlike cr-1 and Δgna-1 strains, the Δgna-1 cr-1 mutant does not produce protoperithecia. The Δgna-1 and cr-1 mutations were synergistic with respect to inappropriate conidiation during growth in submerged culture. Thermotolerance followed the order wild type < Δgna-1 < cr-1 = Δgna-1 cr-1, consistent with a cAMP-dependent process. Taken together, the results suggest that in general, GNA-1 and CR-1 regulate N. crassa growth and development using parallel pathways, while thermotolerance is largely dependent on cAMP.     

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.     

The Rho-GEF Rom2p Localizes to Sites of Polarized Cell Growth and Participates in Cytoskeletal Functions in Saccharomyces cerevisiae

Rom2p is a GDP/GTP exchange factor for Rho1p and Rho2p GTPases; Rho proteins have been implicated in control of actin cytoskeletal rearrangements. ROM2 and RHO2 were identified in a screen for high-copy number suppressors of cik1Δ, a mutant defective in microtubule-based processes in Saccharomyces cerevisiae. A Rom2p::3XHA fusion protein localizes to sites of polarized cell growth, including incipient bud sites, tips of small buds, and tips of mating projections. Disruption of ROM2 results in temperature-sensitive growth defects at 11°C and 37°C. rom2Δ cells exhibit morphological defects. At permissive temperatures, rom2Δ cells often form elongated buds and fail to form normal mating projections after exposure to pheromone; at the restrictive temperature, small budded cells accumulate. High-copy number plasmids containing either ROM2 or RHO2 suppress the temperature-sensitive growth defects of cik1Δ and kar3Δ strains. KAR3 encodes a kinesin-related protein that interacts with Cik1p. Furthermore, rom2Δ strains exhibit increased sensitivity to the microtubule depolymerizing drug benomyl. These results suggest a role for Rom2p in both polarized morphogenesis and functions of the microtubule cytoskeleton.     

Prm1 Functions as a Disulfide-linked Complex in Yeast Mating

Prm1 is a pheromone-induced membrane glycoprotein that promotes plasma membrane fusion in yeast mating pairs. HA-Prm1 migrates at twice its expected molecular weight on non-reducing SDS-PAGE gels and coprecipitates with Prm1-TAP, indicating that Prm1 is a disulfide-linked homodimer. The N terminus of a plasma membrane-localized GFP-Prm1 endocytic mutant projects into the cytoplasm, where it is protected from low pH quenching in live cells and from external protease in spheroplasts. In a revised topological map, Prm1 has four transmembrane domains and two large extracellular loops. Mutation of all four cysteines in the extracellular loops blocked disulfide bond formation and destabilized the Prm1 homodimer without preventing Prm1 transport to contact sites in mating pairs. Cys¹²⁰ in loop 1 and Cys⁵⁴⁵ in loop 2 form disulfide cross-links in the Prm1 homodimer and are required for fusion activity. Cys¹²⁰ lies between a hydrophobic segment formerly thought to be a transmembrane domain and an amphipathic helix. An interaction between either of these regions and the opposing membrane could promote fusion.     

Engineering Neurospora crassa for Improved Cellobiose and Cellobionate Production

We report engineering Neurospora crassa to improve the yield of cellobiose and cellobionate from cellulose. A previously engineered strain of N. crassa (F5) with six of seven β-glucosidase (bgl) genes knocked out was shown to produce cellobiose and cellobionate directly from cellulose without the addition of exogenous cellulases. In this study, the F5 strain was further modified to improve the yield of cellobiose and cellobionate from cellulose by increasing cellulase production and decreasing product consumption. The effects of two catabolite repression genes, cre-1 and ace-1, on cellulase production were investigated. The F5 Δace-1 mutant showed no improvement over the wild type. The F5 Δcre-1 and F5 Δace-1 Δcre-1 strains showed improved cellobiose dehydrogenase and exoglucanase expression. However, this improvement in cellulase expression did not lead to an improvement in cellobiose or cellobionate production. The cellobionate phosphorylase gene (ndvB) was deleted from the genome of F5 Δace-1 Δcre-1 to prevent the consumption of cellobiose and cellobionate. Despite a slightly reduced hydrolysis rate, the F5 Δace-1 Δcre-1 ΔndvB strain converted 75% of the cellulose consumed to the desired products, cellobiose and cellobionate, compared to 18% converted by the strain F5 Δace-1 Δcre-1.     

Role of a mitogen-activated protein kinase pathway during conidial germination and hyphal fusion in Neurospora crassa

Mitogen-activated protein (MAP) kinase signaling pathways are ubiquitous and evolutionarily conserved in eukaryotic organisms. MAP kinase pathways are composed of a MAP kinase, a MAP kinase kinase, and a MAP kinase kinase kinase; activation is regulated by sequential phosphorylation. Components of three MAP kinase pathways have been identified by genome sequence analysis in the filamentous fungus Neurospora crassa. One of the predicted MAP kinases in N. crassa, MAK-2, shows similarity to Fus3p and Kss1p of Saccharomyces cerevisiae, which are involved in sexual reproduction and filamentation, respectively. In this study, we show that an N. crassa mutant disrupted in mak-2 exhibits a pleiotropic phenotype: derepressed conidiation, shortened aerial hyphae, lack of vegetative hyphal fusion, female sterility, and autonomous ascospore lethality. We assessed the phosphorylation of MAK-2 during conidial germination and early colony development. Peak levels of MAK-2 phosphorylation were most closely associated with germ tube elongation, branching, and hyphal fusion events between conidial germlings. A MAP kinase kinase kinase (NRC-1) is the predicted product of N. crassa nrc-1 locus and is a homologue of STE11 in S. cerevisiae. An nrc-1 mutant shares many of the same phenotypic traits as the mak-2 mutant and, in particular, is a hyphal fusion mutant. We show that MAK-2 phosphorylation during early colony development is dependent upon the presence of NRC-1 and postulate that phosphorylation of MAK-2 is required for hyphal fusion events that occur during conidial germination.     

The response regulator RRG-1 functions upstream of a mitogen-activated protein kinase pathway impacting asexual development, female fertility, osmotic stress, and fungicide resistance in Neurospora crassa

Two-component systems, consisting of proteins with histidine kinase and/or response regulator domains, regulate environmental responses in bacteria, Archaea, fungi, slime molds, and plants. Here, we characterize RRG-1, a response regulator protein from the filamentous fungus Neurospora crassa. The cell lysis phenotype of Δrrg-1 mutants is reminiscent of osmotic-sensitive (os) mutants, including nik-1/os-1 (a histidine kinase) and strains defective in components of a mitogen-activated protein kinase (MAPK) pathway: os-4 (MAPK kinase kinase), os-5 (MAPK kinase), and os-2 (MAPK). Similar to os mutants, Δrrg-1 strains are sensitive to hyperosmotic conditions, and they are resistant to the fungicides fludioxonil and iprodione. Like os-5, os-4, and os-2 mutants, but in contrast to nik-1/os-1 strains, Δrrg-1 mutants do not produce female reproductive structures (protoperithecia) when nitrogen starved. OS-2-phosphate levels are elevated in wild-type cells exposed to NaCl or fludioxonil, but they are nearly undetectable in Δrrg-1 strains. OS-2-phosphate levels are also low in Δrrg-1, os-2, and os-4 mutants under nitrogen starvation. Analysis of the rrg-1^D921N allele, mutated in the predicted phosphorylation site, provides support for phosphorylation-dependent and -independent functions for RRG-1. The data indicate that RRG-1 controls vegetative cell integrity, hyperosmotic sensitivity, fungicide resistance, and protoperithecial development through regulation of the OS-4/OS-5/OS-2 MAPK pathway.     

Structure and function of a mating-type gene from the homothallic species Neurospora africana

The homothallic Neurospora species, N. africana, contains sequences that hybridize to the A but not to a mating-type sequences of the heterothallic species N. crassa. In this study, the N. africana mating-type gene, mt A-1, was cloned, sequenced and its function analyzed in N. crassa. Although N. africana does not mate in a heterothallic manner, its mt A-1 gene functions as a mating activator in N. crassa. In addition, the N. africana mt A-1 gene confers mating type-associated vegetative incompatibility in N. crassa. DNA sequence analysis shows that the N. africana mt A-1 open reading frame (ORF) is 93% identical to that of N. crassa mt A-1. The mt A-1 ORF of N. africana contains no stop codons and was detected as a cDNA which is processed in a similar manner to mt A-1 of N. crassa. By DNA blot and orthogonal field agarose gel electrophoretic analysis, it is shown that the composition and location of the mating-type locus and the organization of the mating-type chromosome of N. africana are similar to that of N. crassa.