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.     

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.     

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.     

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.     

The plasma membrane proteins Prm1 and Fig1 ascertain fidelity of membrane fusion during yeast mating

As for most cell-cell fusion events, the molecular details of membrane fusion during yeast mating are poorly understood. The multipass membrane protein Prm1 is the only known component that acts at the step of bilayer fusion. In its absence, mutant mating pairs lyse or arrest in the mating reaction with tightly apposed plasma membranes. We show that deletion of FIG 1, which controls pheromone-induced Ca(2+) influx, yields similar cell fusion defects. Although extracellular Ca(2+) is not required for efficient cell fusion of wild-type cells, cell fusion in prm1 mutant mating pairs is dramatically reduced when Ca(2+) is removed. This enhanced fusion defect is due to lysis. Time-lapse microscopy reveals that fusion and lysis events initiate with identical kinetics, suggesting that both outcomes result from engagement of the fusion machinery. The yeast synaptotagmin orthologue and Ca(2+) binding protein Tcb3 has a role in reducing lysis of prm1 mutants, which opens the possibility that the observed role of Ca(2+) is to engage a wound repair mechanism. Thus, our results suggest that Prm1 and Fig1 have a role in enhancing membrane fusion and maintaining its fidelity. Their absence results in frequent mating pair lysis, which is counteracted by Ca(2+)-dependent membrane repair.     

Endocytosis is essential for pathogenic development in the corn smut fungus Ustilago maydis

It is well established that polarized exocytosis is essential for fungal virulence. By contrast, the contribution of endocytosis is unknown. We made use of a temperature-sensitive mutant in the endosomal target soluble N-ethylmaleimide-sensitive factor attachment protein receptor Yup1 and demonstrate that endocytosis in Ustilago maydis is essential for the initial steps of pathogenic development, including pheromone perception and cell-cell fusion. Furthermore, spore formation and germination were drastically reduced, whereas colonization of the plant was only slightly inhibited. The function of endocytosis in the recognition of mating pheromone through the G protein-coupled pheromone receptor Pra1 was analyzed in greater detail. Biologically active Pra1-green fluorescent protein localizes to the plasma membrane and is constitutively endocytosed. Yup1(ts) mutants that are blocked in the fusion of endocytic transport vesicles with early endosomes are impaired in pheromone perception and conjugation hyphae formation. This is attributable to an accumulation of Pra1-carrying endocytic vesicles in the cytoplasm and the depletion of the receptor from the membrane. Consistently, strong Pra1 expression rescues the signaling defects in endocytosis mutants, but subsequent cell fusion is still impaired. Thus, we conclude that endocytosis is essential for recognition of the partner at the beginning of the pathogenic program but has additional roles in mating as well as spore formation and germination.     

The Saccharomyces cerevisiae PRM1 Homolog in Neurospora crassa Is Involved in Vegetative and Sexual Cell Fusion Events but Also Has Postfertilization Functions

Cell–cell fusion is essential for a variety of developmental steps in many eukaryotic organisms, during both fertilization and vegetative cell growth. Although the molecular mechanisms associated with intracellular membrane fusion are well characterized, the molecular mechanisms of plasma membrane merger between cells are poorly understood. In the filamentous fungus Neurospora crassa, cell fusion events occur during both vegetative and sexual stages of its life cycle, thus making it an attractive model for studying the molecular basis of cell fusion during vegetative growth vs. sexual reproduction. In the unicellular yeast Saccharomyces cerevisiae, one of the few proteins implicated in plasma membrane merger during mating is Prm1p; prm1Δ mutants show an ~50% reduction in mating cell fusion. Here we report on the role of the PRM1 homolog in N. crassa. N. crassa strains with deletions of a Prm1-like gene (Prm1) showed an ~50% reduction in both vegetative and sexual cell fusion events, suggesting that PRM1 is part of the general cell fusion machinery. However, unlike S. cerevisiae, N. crassa strains carrying a Prm1 deletion exhibited complete sterility as either a male or female mating partner, a phenotype that was not complemented in a heterokaryon with wild type (WT). Crosses with ΔPrm1 strains were blocked early in sexual development, well before development of ascogenous hyphae. The ΔPrm1 sexual defect in N. crassa was not suppressed by mutations in Sad-1, which is required for meiotic silencing of unpaired DNA (MSUD). However, mutations in Sad-1 increased the number of progeny obtained in crosses with a ΔPrm1 (Prm1-gfp) complemented strain. These data indicate multiple roles for PRM1 during sexual development.     

Effects of singlet oxygen on membrane sterols in the yeast Saccharomyces cerevisiae.

Photodynamic treatment of the yeast Saccharomyces cerevisiae with the singlet oxygen sensitizer toluidine blue and visible light leads to rapid oxidation of ergosterol and accumulation of oxidized ergosterol derivatives in the plasma membrane. The predominant oxidation product accumulated was identified as 5alpha, 6alpha-epoxy-(22E)-ergosta-8,22-dien-3beta,7a lpha-diol (8-DED). 9(11)-dehydroergosterol (DHE) was identified as a minor oxidation product. In heat inactivated cells ergosterol is photooxidized to ergosterol epidioxide (EEP) and DHE. Disrupted cell preparations of S. cerevisiae convert EEP to 8-DED, and this activity is abolished in a boiled control indicating the presence of a membrane associated enzyme with an EEP isomerase activity. Yeast selectively mobilizes ergosterol from the intracellular sterol ester pool to replenish the level of free ergosterol in the plasma membrane during singlet oxygen oxidation. The following reaction pathway is proposed: singlet oxygen-mediated oxidation of ergosterol leads to mainly the formation of EEP, which is enzymatically rearranged to 8-DED. Ergosterol 7-hydroperoxide, a known minor product of the reaction of singlet oxygen with ergosterol, is formed at a much lower rate and decomposes to give DHE. Changes of physical properties of the plasma membrane are induced by depletion of ergosterol and accumulation of polar derivatives. Subsequent permeation of photosensitizer through the plasma membrane into the cell leads to events including impairment of mitochondrial function and cell inactivation.     

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.     

Effect of culture conditions on ergosterol as an indicator of biomass in the aquatic hyphomycetes

Ergosterol is a membrane component specific to fungi that can be used to estimate fungal biomass using appropriate factors of conversion. Our objectives were to determine the limits of use of ergosterol content as a measure of biomass for aquatic hyphomycetes, and to evaluate a previously established ergosterol-to-biomass conversion factor. We varied inoculum quality, growth medium, and degree of shaking of four aquatic hyphomycete species. In cultures inoculated with homogenized mycelium, we found a significant effect of shaking condition and culture age on ergosterol content. In liquid cultures with defined medium, ergosterol content reached 10 to 11 microg/mg of mycelium (dry mass) and varied by factors of 2.2 during exponential growth and 1.3 during stationary phase. The increase in ergosterol content during exponential phase could be attributed, at least in part, to rapid depletion of glucose. Oxygen availability to internal hyphae within the mycelial mass is also responsible for the differences found between culture conditions. Ergosterol concentration ranged from 0.8 to 1.6 microg/mg in static cultures inoculated with agar plugs. Ergosterol content varied by a factor of 4 in two media of different richnesses. For different combinations of these parameters, strong (r(2) = 0.83 to 0.98) and highly significant (P < 0.001) linear relationships between ergosterol and mycelial dry mass (up to 110 mg) were observed. Overall, the ergosterol content varied by a factor of 14 (0.8 to 11 mg/g). These results suggest that care must be taken when the ergosterol content is used to compare data generated in different field environments.     

Raft partitioning of the yeast uracil permease during trafficking along the endocytic pathway

Lipid rafts, formed by the lateral association of sphingolipids and cholesterol in the external membrane leaflet, have been implicated in membrane traffic and cell signaling in mammalian cells. Yeast plasma membranes were also recently shown to contain lipid raft microdomains consisting of sphingolipids and ergosterol, and containing several plasma membrane proteins, including Gas1p, a GPI-anchored protein, and the [H⁺] ATPase Pma1p. In this study, we investigated whether lipid rafts were involved in the intracellular trafficking of a yeast transporter, uracil permease, which undergoes ubiquitin-dependent endocytosis. Regardless of its ubiquitination status, uracil permease was found to be associated with rafts in the plasma membrane. The expression of Fur4p in lcb1–100 cells, deficient in the first enzyme of sphingolipid synthesis, impaired the association of Fur4p with detergent-resistant fractions. When targeted to endocytic compartments, uracil permease appeared to be progressively transferred to detergent-soluble fractions, suggesting that the lipid environment might change between plasma membrane and endosomes. Consistent with this hypothesis, the wild-type form of the v-SNARE Snc1p, which is known to cycle between the plasma membrane and endosomal compartments, was recovered in both detergent-resistant and detergent-soluble fractions. In contrast, a variant Snc1p that accumulates at the plasma membrane was recovered exclusively in detergent-resistant fractions .     

Molecular basis for fungicidal action of neothyonidioside, a triterpene glycoside from the sea cucumber, Australostichopus mollis

Neothyonidioside is a triterpene glycoside (TG) isolated from the sea cucumber, Australostichopus mollis, that is potently cytotoxic to S. cerevisiae, but does not permeabilize cellular membranes. We mutagenized S. cerevisiae and isolated a neothionidioside-resistant (neo(R)) strain. Using synthetic genetic array mapping and sequencing, we identified NCP1 as the resistance locus. Quantitative HPLC revealed that neo(R)/ncp1 mutants have reduced ergosterol content. Ergosterol added to growth media reversed toxicity, demonstrating that neothionidioside binds directly to ergosterol, similar to the polyene natamycin. Ergosterol synthesis inhibitors ketoconazole and atorvastatin conferred resistance to neothionidioside in a dose-dependent manner showing that a threshold ergosterol concentration is required for toxicity. A genome-wide screen of deletion mutants against neothionidioside revealed hypersensitivity of many of the component genes in the ESCRT complexes relating to multivesicular body formation. Confocal microscopy of cells stained with a vital dye showed blockage at this step. Thus, we propose neothionidioside may affect membrane curvature and fusion capability in the endosome-vacuole pathway.     

Fus1p interacts with components of the Hog1p mitogen-activated protein kinase and Cdc42p morphogenesis signaling pathways to control cell fusion during yeast mating

Cell fusion in the budding yeast Saccharomyces cerevisiae is a temporally and spatially regulated process that involves degradation of the septum, which is composed of cell wall material, and occurs between conjugating cells within a prezygote, followed by plasma membrane fusion. The plasma membrane protein Fus1p is known to be required for septum degradation during cell fusion, yet its role at the molecular level is not understood. We identified Sho1p, an osmosensor for the HOG MAPK pathway, as a binding partner for Fus1 in a two-hybrid screen. The Sho1p-Fus1p interaction occurs directly and is mediated through the Sho1p-SH3 domain and a proline-rich peptide ligand on the Fus1p COOH-terminal cytoplasmic region. The cell fusion defect associated with fus1Delta mutants is suppressed by a sho1Delta deletion allele, suggesting that Fus1p negatively regulates Sho1p signaling to ensure efficient cell fusion. A two-hybrid matrix containing fusion proteins and pheromone response pathway signaling molecules reveals that Fus1p may participate in a complex network of interactions. In particular, the Fus1p cytoplasmic domain interacts with Chs5p, a protein required for secretion of specialized Chs3p-containing vesicles during bud development, and chs5Delta mutants were defective in cell surface localization of Fus1p. The Fus1p cytoplasmic domain also interacts with the activated GTP-bound form of Cdc42p and the Fus1p-SH3 domain interacts with Bni1p, a yeast formin that participates in cell fusion and controls the assembly of actin cables to polarize secretion in response to Cdc42p signaling. Taken together, our results suggest that Fus1p acts as a scaffold for the assembly of a cell surface complex involved in polarized secretion of septum-degrading enzymes and inhibition of HOG pathway signaling to promote cell fusion.     

Specific perception of ergosterol by plant cells

Ergosterol (a fungal membrane component) induced modification of proton fluxes and membrane hyperpolarization in motor cells of Mimosa pudica. These reactions appear specific since they were not induced by the other sterols tested. A specific desensitization was observed, since cells did not react to a second ergosterol application. Exposed at first to other sterols, cells remained reactive to ergosterol. Comparatively, chitosan (a fungal wall component with known elicitor properties) triggered a membrane depolarization and also induced specific desensitization. This comparative study shows that ergosterol and chitosan are distinctly perceived by plant cells and induced different early events at the plasma membrane level.     

Asg7p-Ste3p inhibition of pheromone signaling: regulation of the zygotic transition to vegetative growth

The inappropriate expression of the a-factor pheromone receptor (Ste3p) in the MATa cell leads to a striking inhibition of the yeast pheromone response, the result of a functional interaction between Ste3p and some MATa-specific protein. The present work identifies this protein as Asg7p. Normally, expression of Ste3p and Asg7p is limited to distinct haploid mating types, Ste3p to MATalpha cells and Asg7p to MATa cells. Artificial coexpression of the two in the same cell, either a or alpha, leads to dramatic inhibition of the pheromone response. Ste3p-Asg7p coexpression also perturbs the membrane trafficking of Ste3p: Ste3p turnover is slowed, a result of an Asg7p-mediated retardation of the secretory delivery of the newly synthesized receptor to the plasma membrane. However, in the absence of ectopic Ste3p expression, the asg7Delta mutation is without consequence either for pheromone signaling or overall mating efficiency of a cells. Indeed, the sole phenotype that can be assigned to MATa asg7Delta cells is observed following zygotic fusion to its alpha mating partner. Though formed at wild-type efficiency, zygotes from these pairings are morphologically abnormal. The pattern of growth is deranged: emergence of the first mitotic bud is delayed, and, in its place, growth is apparently diverted into a novel structure superficially resembling the polarized mating projection characteristic of haploid cells responding to pheromone. Together these results suggest a mechanism in which, following the zygotic fusion event, Ste3p and Asg7p gain access to one another and together act to repress the pheromone response, promoting the transition of the new diploid cell to vegetative growth.     

Fig1 facilitates calcium influx and localizes to membranes destined to undergo fusion during mating in Candida albicans

Few mating-regulated genes have been characterized in Candida albicans. C. albicans FIG1 (CaFIG1) is a fungus-specific and mating-induced gene encoding a putative 4-transmembrane domain protein that shares sequence similarities with members of the claudin superfamily. In Saccharomyces cerevisiae, Fig1 is required for shmoo fusion and is upregulated in response to mating pheromones. Expression of CaFIG1 was also strongly activated in the presence of cells of the opposite mating type. CaFig1-green fluorescent protein (GFP) was visible only during the mating response, when it localized predominantly to the plasma membrane and perinuclear zone in mating projections and daughter cells. At the plasma membrane, CaFig1-GFP was visualized as discontinuous zones, but the distribution of perinuclear CaFig1-GFP was homogeneous. Exposure to pheromone induced a 5-fold increase in Ca(2+) uptake in mating-competent opaque cells. Uptake was reduced substantially in the fig1Δ null mutant. CaFig1 is therefore involved in Ca(2+) influx and localizes to membranes that are destined to undergo fusion during mating.     

Plasma membrane polarization during mating in yeast cells

The yeast mating cell provides a simple paradigm for analyzing mechanisms underlying the generation of surface polarity. Endocytic recycling and slow diffusion on the plasma membrane were shown to facilitate polarized surface distribution of Snc1p (Valdez-Taubas, J., and H.R. Pelham. 2003. Curr. Biol. 13:1636-1640). Here, we found that polarization of Fus1p, a raft-associated type I transmembrane protein involved in cell fusion, does not depend on endocytosis. Instead, Fus1p localization to the tip of the mating projection was determined by its cytosolic domain, which binds to peripheral proteins involved in mating tip polarization. Furthermore, we provide evidence that the lipid bilayer at the mating projection is more condensed than the plasma membrane enclosing the cell body, and that sphingolipids are required for this lipid organization.     

Sphingolipids, cholesterol, and HIV-1: A paradigm in viral fusion

Our previous studies show that the depletion of cholesterol or sphingolipids (raft-associated lipids) from receptor-bearing adherent cell lines blocks HIV-1 entry and HIV-1 Env-mediated membrane fusion. Here we have evaluated the mechanism(s) by which these lipids contribute to the HIV-1 Env-mediated membrane fusion. We report the following: (1) GSL depletion from a suspension T lymphocyte cell line (Sup-T1) reduced subsequent fusion with HIV-1IIIB-expressing cells by 70%. (2) Cholesterol depletion from NIH3T3 cells bearing HIV-1 receptors (NIH3T3CD4R5/NIH3T3CD4X4) did not impair subsequent fusion with HeLa cells expressing the corresponding HIV-1 Envs. In contrast GSL depletion from these targets reduced fusion by 50% suggesting that GSL facilitate fusion in different ways. (3) GSL-deficient GM95 cells bearing high receptors fused with HIV-1 Env-expressing cells at 37°C with kinetics similar to that of GSL + NIH3T3 targets. Based on these observations, we propose that the plasma membrane cholesterol is required to maintain the integrity of receptor pools whereas GSLs are involved in stabilizing the coupling of inter-receptor pools.     

Reorganization of membrane cholesterol during membrane fusion in myogenesis in vitro: a study using the filipin-cholesterol complex

Using filipin and freeze-fracture electron microscopy, we examined the distribution of membrane cholesterol during the fusion of myoblasts in vitro. The early stages of fusion were characterized by the depletion of cholesterol from the membrane apposition sites, at which the plasma membranes of two adjacent cells were in close contact. At first, filipin-cholesterol complexes were absent from the plasma membrane of one cell only and were distributed homogeneously on the membrane of the other cell. Eventually, both of the closely apposed membranes became almost completely free the filipin-cholesterol complexes. Membrane fusion took place at several points within the filipin-cholesterol complex-free areas. In later stages, the cytoplasms of the fusing cells became confluent by fenestration of the plasma membranes formed with the filipin-cholesterol complex-free regions. Our observations suggest that membrane cholesterol is reorganized at these fusion sites and that fusion initiated by the juxtaposition of the cholesterol-free areas of each plasma membrane of the adjacent cells.