Enhanced Fusion Pore Expansion Mediated by the Trans-Acting Endodomain of the Reovirus FAST Proteins

The reovirus fusion-associated small transmembrane (FAST) proteins are virus-encoded membrane fusion proteins that function as dedicated cell–cell fusogens. The topology of these small, single-pass membrane proteins orients the majority of the protein on the distal side of the membrane (i.e., inside the cell). We now show that ectopic expression of the endodomains of the p10, p14, and p15 FAST proteins enhances syncytiogenesis induced by the full-length FAST proteins, both homotypically and heterotypically. Results further indicate that the 68-residue cytoplasmic endodomain of the p14 FAST protein (1) is endogenously generated from full-length p14 protein expressed in virus-infected or transfected cells; (2) enhances syncytiogenesis subsequent to stable pore formation; (3) increases the syncytiogenic activity of heterologous fusion proteins, including the differentiation-dependent fusion of murine myoblasts; (4) exerts its enhancing activity from the cytosol, independent of direct interactions with either the fusogen or the membranes being fused; and (5) contains several regions with protein–protein interaction motifs that influence enhancing activity. We propose that the unique evolution of the FAST proteins as virus-encoded cellular fusogens has allowed them to generate a trans-acting, soluble endodomain peptide to harness a cellular pathway or process involved in the poorly understood process that facilitates the transition from microfusion pores to macrofusion and syncytiogenesis.     

A virus-encoded cell-cell fusion machine dependent on surrogate adhesins

The reovirus fusion-associated small transmembrane (FAST) proteins function as virus-encoded cellular fusogens, mediating efficient cell-cell rather than virus-cell membrane fusion. With ectodomains of only approximately 20-40 residues, it is unclear how such diminutive viral fusion proteins mediate the initial stages (i.e. membrane contact and close membrane apposition) of the fusion reaction that precede actual membrane merger. We now show that the FAST proteins lack specific receptor-binding activity, and in their natural biological context of promoting cell-cell fusion, rely on cadherins to promote close membrane apposition. The FAST proteins, however, are not specifically reliant on cadherin engagement to mediate membrane apposition as indicated by their ability to efficiently utilize other adhesins in the fusion reaction. Results further indicate that surrogate adhesion proteins that bridge membranes as close as 13 nm apart enhance FAST protein-induced cell-cell fusion, but active actin remodelling is required for maximal fusion activity. The FAST proteins are the first example of membrane fusion proteins that have specifically evolved to function as opportunistic fusogens, designed to exploit and convert naturally occurring adhesion sites into fusion sites. The capacity of surrogate, non-cognate adhesins and active actin remodelling to enhance the cell-cell fusion activity of the FAST proteins are features perfectly suited to the structural and functional evolution of these fusogens as the minimal fusion component of a virus-encoded cellular fusion machine. These results also provide a basis for reconciling the rudimentary structure of the FAST proteins with their capacity to fuse cellular membranes.     

Features of a Spatially Constrained Cystine Loop in the p10 FAST Protein Ectodomain Define a New Class of Viral Fusion Peptides

The reovirus fusion-associated small transmembrane (FAST) proteins are the smallest known viral membrane fusion proteins. With ectodomains of only ∼20–40 residues, it is unclear how such diminutive fusion proteins can mediate cell-cell fusion and syncytium formation. Contained within the 40-residue ectodomain of the p10 FAST protein resides an 11-residue sequence of moderately apolar residues, termed the hydrophobic patch (HP). Previous studies indicate the p10 HP shares operational features with the fusion peptide motifs found within the enveloped virus membrane fusion proteins. Using biotinylation assays, we now report that two highly conserved cysteine residues flanking the p10 HP form an essential intramolecular disulfide bond to create a cystine loop. Mutagenic analyses revealed that both formation of the cystine loop and p10 membrane fusion activity are highly sensitive to changes in the size and spatial arrangement of amino acids within the loop. The p10 cystine loop may therefore function as a cystine noose, where fusion peptide activity is dependent on structural constraints within the noose that force solvent exposure of key hydrophobic residues. Moreover, inhibitors of cell surface thioreductase activity indicate that disruption of the disulfide bridge is important for p10-mediated membrane fusion. This is the first example of a viral fusion peptide composed of a small, spatially constrained cystine loop whose function is dependent on altered loop formation, and it suggests the p10 cystine loop represents a new class of viral fusion peptides.     

The p14 fusion-associated small transmembrane (FAST) protein effects membrane fusion from a subset of membrane microdomains

The reovirus fusion-associated small transmembrane (FAST) proteins are a unique family of viral membrane fusion proteins. These nonstructural viral proteins induce efficient cell-cell rather than virus-cell membrane fusion. We analyzed the lipid environment in which the reptilian reovirus p14 FAST protein resides to determine the influence of the cell membrane on the fusion activity of the FAST proteins. Topographical mapping of the surface of fusogenic p14-containing liposomes by atomic force microscopy under aqueous conditions revealed that p14 resides almost exclusively in thickened membrane microdomains. In transfected cells, p14 was found in both Lubrol WX- and Triton X-100-resistant membrane complexes. Cholesterol depletion of donor cell membranes led to preferential disruption of p14 association with Lubrol WX (but not Triton X-100)-resistant membranes and decreased cell-cell fusion activity, both of which were reversed upon subsequent cholesterol repletion. Furthermore, co-patching analysis by fluorescence microscopy indicated that p14 did not co-localize with classical lipid-anchored raft markers. These data suggest that the p14 FAST protein associates with heterogeneous membrane microdomains, a distinct subset of which is defined by cholesterol-dependent Lubrol WX resistance and which may be more relevant to the membrane fusion process.     

Helix-destabilizing, beta-branched, and polar residues in the baboon reovirus p15 transmembrane domain influence the modularity of FAST proteins

The fusogenic reoviruses induce syncytium formation using the fusion-associated small transmembrane (FAST) proteins. A recent study indicated the p14 FAST protein transmembrane domain (TMD) can be functionally replaced by the TMDs of the other FAST proteins but not by heterologous TMDs, suggesting that the FAST protein TMDs are modular fusion units. We now show that the p15 FAST protein is also a modular fusogen, as indicated by the functional replacement of the p15 ectodomain with the corresponding domain from the p14 FAST protein. Paradoxically, the p15 TMD is not interchangeable with the TMDs of the other FAST proteins, implying that unique attributes of the p15 TMD are required when this fusion module is functioning in the context of the p15 ecto- and/or endodomain. A series of point substitutions, truncations, and reextensions were created in the p15 TMD to define features that are specific to the functioning of the p15 TMD. Removal of only one or two residues from the N terminus or four residues from the C terminus of the p15 TMD eliminated membrane fusion activity, and there was a direct correlation between the fusion-promoting function of the p15 TMD and the presence of N-terminal, hydrophobic β-branched residues. Substitution of the glycine residues and triserine motif present in the p15 TMD also impaired or eliminated the fusion-promoting activity of the p15 TMD. The ability of the p15 TMD to function in an ecto- and endodomain-specific context is therefore influenced by stringent sequence requirements that reflect the importance of TMD polar residues and helix-destabilizing residues.     

Reptilian reovirus utilizes a small type III protein with an external myristylated amino terminus to mediate cell-cell fusion

Reptilian reovirus is one of a limited number of nonenveloped viruses that are capable of inducing cell-cell fusion. A small, hydrophobic, basic, 125-amino-acid fusion protein encoded by the first open reading frame of a bicistronic viral mRNA is responsible for this fusion activity. Sequence comparisons to previously characterized reovirus fusion proteins indicated that p14 represents a new member of the fusion-associated small transmembrane (FAST) protein family. Topological analysis revealed that p14 is a representative of a minor subset of integral membrane proteins, the type III proteins N(exoplasmic)/C(cytoplasmic) (N(exo)/C(cyt)), that lack a cleavable signal sequence and use an internal reverse signal-anchor sequence to direct membrane insertion and protein topology. This topology results in the unexpected, cotranslational translocation of the essential myristylated N-terminal domain of p14 across the cell membrane. The topology and structural motifs present in this novel reovirus membrane fusion protein further accentuate the diversity and unusual properties of the FAST protein family and clearly indicate that the FAST proteins represent a third distinct class of viral membrane fusion proteins.     

Multifaceted Sequence-Dependent and -Independent Roles for Reovirus FAST Protein Cytoplasmic Tails in Fusion Pore Formation and Syncytiogenesis

Fusogenic reoviruses utilize the FAST proteins, a novel family of nonstructural viral membrane fusion proteins, to induce cell-cell fusion and syncytium formation. Unlike the paradigmatic enveloped virus fusion proteins, the FAST proteins position the majority of their mass within and internal to the membrane in which they reside, resulting in extended C-terminal cytoplasmic tails (CTs). Using tail truncations, we demonstrate that the last 8 residues of the 36-residue CT of the avian reovirus p10 FAST protein and the last 20 residues of the 68-residue CT of the reptilian reovirus p14 FAST protein enhance, but are not required for, pore expansion and syncytium formation. Further truncations indicate that the membrane-distal 12 residues of the p10 and 47 residues of the p14 CTs are essential for pore formation and that a residual tail of 21 to 24 residues that includes a conserved, membrane-proximal polybasic region present in all FAST proteins is insufficient to maintain FAST protein fusion activity. Unexpectedly, a reextension of the tail-truncated, nonfusogenic p10 and p14 constructs with scrambled versions of the deleted sequences restored pore formation and syncytiogenesis, while reextensions with heterologous sequences partially restored pore formation but failed to rescue syncytiogenesis. The membrane-distal regions of the FAST protein CTs therefore exert multiple effects on the membrane fusion reaction, serving in both sequence-dependent and sequence-independent manners as positive effectors of pore formation, pore expansion, and syncytiogenesis.The only examples of nonenveloped viruses that induce cell-cell fusion and syncytium formation occur within the family Orthoreoviridae, an extremely diverse group of viruses containing segmented double-stranded RNA genomes (9). In recent years, the viral proteins responsible for the syncytiogenic phenotype of the fusogenic orthoreoviruses and aquareoviruses have been identified and characterized (14, 18, 41, 46). These fusion-associated small transmembrane (FAST) proteins define a new family of viral fusogens with several unique biological and biophysical properties. Unlike the well-characterized enveloped virus fusion proteins, reovirus FAST proteins are nonstructural viral proteins and are therefore not involved in mediating virus-cell fusion and virus entry (18, 21, 46). The FAST proteins are instead dedicated to inducing cell-cell fusion and syncytium formation following their expression and trafficking to the plasma membrane of virus-infected or transfected cells (14, 17, 46). Data from previously reported studies also suggest that the FAST proteins serve as virulence factors for the fusogenic reoviruses, promoting virus dissemination and increased tissue destruction (6, 43). How this atypical family of viral fusogens functions to mediate cell-cell membrane fusion remains unclear.The unusual biological role of the FAST proteins as nonstructural, virus-encoded, “cellular” fusogens is embodied in structural features that clearly distinguish the FAST proteins from the membrane fusion proteins of enveloped viruses. There are currently four distinct members of the FAST protein family, named according to their molecular masses: the homologous p10 proteins of avian reovirus (ARV) and Nelson Bay reovirus and the unrelated p14, p15, and p22 proteins of reptilian reovirus (RRV), baboon reovirus, and Atlantic salmon aquareovirus, respectively (14, 18, 41, 46). These proteins are the smallest known fusogens, ranging from 95 to 198 amino acids in size, and assume an asymmetric topology in the plasma membrane, with a single transmembrane domain that separates small N-terminal ectodomains of ∼20 to 41 residues from equal-sized or considerably larger C-terminal endodomains of ∼36 to 141 residues (Fig. ​(Fig.1A).1A). A number of structural motifs in both the ecto- and endodomains of the FAST proteins have been identified, including sites of acylation, hydrophobic patches, a membrane-proximal polybasic region, and regions rich in proline, cysteine, or arginine, proline, and histidine. Each of the FAST proteins has its own signature repertoire and arrangement of these motifs. Determining how these various motifs contribute to the fusogenic activity of the FAST proteins remains an area of active investigation.Open in a separate windowFIG. 1.ARV p10 and RRV p14 FAST protein topologies and tail truncations. (A) Diagrammatic representation of the p10 and p14 FAST proteins showing their topology in the plasma membrane. Both are single-pass transmembrane proteins with N-terminal ectodomains on the surface of cells and C-terminal endodomains in the cytoplasm. Structural motifs include hydrophobic patches (HP), polybasic motifs (PB), fatty acid modifications (indicated by squiggly lines) that are either the N-terminal myristoylation or palmitoylation of a dicysteine motif (CC), and a polyproline motif (PP). The total number of residues in each protein is indicated by the numbers. (B) The amino acid sequences of the p10 and p14 endodomains are shown, along with the motifs described above. Progressive truncations of the CTs were constructed (arrows), with the numbers indicating the last amino acid present in the full-length proteins or each truncation.Numerous studies of diverse fusion processes define five general steps of the pathway for membrane fusion and syncytium formation: membrane binding, close membrane apposition, hemifusion (i.e., the mixing of the outer leaflets of the two bilayers), stable pore formation, and pore expansion (12, 13, 44). The well-characterized enveloped virus fusion proteins utilize extensive structural rearrangement of their complex ectodomains to provide mechanical energy to draw membranes into close proximity and promote membrane merger (21, 53). The limited size of the FAST protein ectodomains precludes such a mechanical model for membrane fusion, necessitating the development of alternate models to explain how the diminutive FAST proteins breach the thermodynamic barriers that prevent the spontaneous merger of biological membranes. The FAST proteins are both necessary and sufficient to mediate membrane fusion (51). However, data from recent studies indicate that for maximal cell fusion activity, the FAST proteins rely on surrogate adhesins to mediate close membrane apposition (42). Data from recent studies also indicate that a small percentage of the p14 FAST protein expressed in virus-infected or transfected cells is proteolytically processed to generate a bioactive, soluble endodomain that recruits cellular pathways to drive the expansion of stable fusion pores into the extended fusion apertures needed for syncytium formation (50). The FAST proteins therefore utilize accessory proteins to mediate the prefusion (membrane binding and apposition) and postfusion (pore expansion) stages of syncytiogenesis, retaining within their rudimentary structures all that is required to mediate the actual process of membrane merger. This subdivision of the multistep process of syncytium formation is reflected in, and is perfectly suited to, the evolution of the FAST proteins as virus-encoded cellular fusogens.The small size of the FAST protein ectodomains and their donor membrane-focused topology contrast markedly with enveloped virus fusion proteins that position the majority of their mass external to the membrane. While the complex ectodomains of the enveloped virus fusion proteins clearly play an essential role in the fusion reaction, the involvement of their cytoplasmic tails (CTs) is far less certain, and no consistent picture of the role of these C-terminal tails has emerged. The CTs of many enveloped viral fusion proteins, including baculovirus (31), severe acute respiratory syndrome coronavirus (5), vesicular stomatitis virus (36), parainfluenza virus type 2 (56), and influenza A virus subtype H3 (10), play no role in the membrane fusion reaction. Of the fusion protein tails that do modulate the fusion reaction, the majority serve inhibitory roles, including the F proteins of measles virus and parainfluenza virus type 5 SER (7, 45, 52), glycoprotein B from several herpesviruses (22, 24, 28), and the fusion proteins of numerous retroviruses (1, 8, 30, 32, 34, 47, 48). These inhibitory cytoplasmic domains alter the conformation of the fusion protein ectodomains, thereby coupling virion maturation to fusion competence (1, 2, 35, 52, 54). In the few cases where extensive tail truncations adversely affect fusion, these truncations generally decrease but do not eliminate syncytiogenesis, and it is the membrane-proximal portion of the tail that promotes pore formation or pore expansion (20, 25, 26, 32).Since the FAST proteins are nonstructural viral proteins, their CTs (also referred to as endodomains) are not required to suppress fusion activity until after virus particle assembly. At the same time, the disproportionate size of their endodomains strongly suggests that these CTs play an important role in membrane fusion activity. Although one such role of the p14 CT is the generation of a soluble endodomain that recruits cellular factors involved in pore expansion, the majority of p14 is not proteolytically processed, suggesting that FAST protein CTs may serve additional roles as components of the intact protein (50). We now show that C-terminal truncations of the p10 and p14 FAST proteins reduced and eventually eliminated cell-cell fusion. Fluorescence-based pore formation assays coupled with tail reextension studies further revealed that FAST protein CTs drive fusion pore formation and expansion in both sequence-dependent and sequence-independent manners. The membrane-distal regions of FAST protein CTs therefore exert multiple effects on the mechanism of membrane fusion.     

Extensive syncytium formation mediated by the reovirus FAST proteins triggers apoptosis-induced membrane instability

The fusion-associated small transmembrane (FAST) proteins of the fusogenic reoviruses are the only known examples of membrane fusion proteins encoded by non-enveloped viruses. While the involvement of the FAST proteins in mediating extensive syncytium formation in virus-infected and -transfected cells is well established, the nature of the fusion reaction and the role of cell-cell fusion in the virus replication cycle remain unclear. To address these issues, we analyzed the syncytial phenotype induced by four different FAST proteins: the avian and Nelson Bay reovirus p10, reptilian reovirus p14, and baboon reovirus p15 FAST proteins. Results indicate that FAST protein-mediated cell-cell fusion is a relatively non-leaky process, as demonstrated by the absence of significant [3H]uridine release from cells undergoing fusion and by the resistance of these cells to treatment with hygromycin B, a membrane-impermeable translation inhibitor. However, diminished membrane integrity occurred subsequent to extensive syncytium formation and was associated with DNA fragmentation and chromatin condensation, indicating that extensive cell-cell fusion activates apoptotic signaling cascades. Inhibiting effector caspase activation or ablating the extent of syncytium formation, either by partial deletion of the avian reovirus p10 ecto-domain or by antibody inhibition of p14-mediated cell-cell fusion, all resulted in reduced membrane permeability changes. These observations suggest that the FAST proteins do not possess intrinsic membrane-lytic activity. Rather, extensive FAST protein-induced syncytium formation triggers an apoptotic response that contributes to altered membrane integrity. We propose that the FAST proteins have evolved to serve a dual role in the replication cycle of these fusogenic non-enveloped viruses, with non-leaky cell-cell fusion initially promoting localized cell-cell transmission of the infection followed by enhanced progeny virus release from apoptotic syncytia and systemic dissemination of the infection.     

Myristoylation, a protruding loop, and structural plasticity are essential features of a nonenveloped virus fusion peptide motif

Members of the fusion-associated small transmembrane (FAST) protein family are a distinct class of membrane fusion proteins encoded by nonenveloped fusogenic reoviruses. The 125-residue p14 FAST protein of reptilian reovirus has an approximately 38-residue myristoylated N-terminal ectodomain containing a moderately apolar N-proximal region, termed the hydrophobic patch. Mutagenic analysis indicated sequence-specific elements in the N-proximal portion of the p14 hydrophobic patch affected cell-cell fusion activity, independent of overall effects on the relative hydrophobicity of the motif. Circular dichroism (CD) of a myristoylated peptide representing the majority of the p14 ectodomain suggested this region is mostly disordered in solution but assumes increased structure in an apolar environment. From NMR spectroscopic data and simulated annealing, the soluble nonmyristoylated p14 ectodomain peptide consists of an N-proximal extended loop flanked by two proline hinges. The remaining two-thirds of the ectodomain peptide structure is disordered, consistent with predictions based on CD spectra of the myristoylated peptide. The myristoylated p14 ectodomain peptide, but not a nonmyristoylated version of the same peptide nor a myristoylated scrambled peptide, mediated extensive lipid mixing in a liposome fusion assay. Based on the lipid mixing activity, structural plasticity, environmentally induced conformational changes, and kinked structures predicted for the p14 ectodomain and hydrophobic patch (all features associated with fusion peptides), we propose that the majority of the p14 ectodomain is composed of a fusion peptide motif, the first such motif dependent on myristoylation for membrane fusion activity.     

The p14 FAST protein of reptilian reovirus increases vesicular stomatitis virus neuropathogenesis

The fusogenic orthoreoviruses express nonstructural fusion-associated small transmembrane (FAST) proteins that induce cell-cell fusion and syncytium formation. It has been speculated that the FAST proteins may serve as virulence factors by promoting virus dissemination and increased or altered cytopathology. To directly test this hypothesis, the gene encoding the p14 FAST protein of reptilian reovirus was inserted into the genome of a heterologous virus that does not naturally form syncytia, vesicular stomatitis virus (VSV). Expression of the p14 FAST protein by the VSV/FAST recombinant gave the virus a highly fusogenic phenotype in cell culture. The growth of this recombinant fusogenic VSV strain was unaltered in vitro but was significantly enhanced in vivo. The VSV/FAST recombinant consistently generated higher titers of virus in the brains of BALB/c mice after intranasal or intravenous infection compared to the parental VSV/green fluorescent protein (GFP) strain that expresses GFP in place of p14. The VSV/FAST recombinant also resulted in an increased incidence of hind-limb paralysis, it infected a larger volume of brain tissue, and it induced more extensive neuropathology, thus leading to a lower maximum tolerable dose than that for the VSV/GFP parental virus. In contrast, an interferon-inducing mutant of VSV expressing p14 was still attenuated, indicating that this interferon-inducing phenotype is dominant to the fusogenic properties conveyed by the FAST protein. Based on this evidence, we conclude that the reovirus p14 FAST protein can function as a bona fide virulence factor.     

Unusual topological arrangement of structural motifs in the baboon reovirus fusion-associated small transmembrane protein

Select members of the Reoviridae are the only nonenveloped viruses known to induce syncytium formation. The fusogenic orthoreoviruses accomplish cell-cell fusion through a distinct class of membrane fusion-inducing proteins referred to as the fusion-associated small transmembrane (FAST) proteins. The p15 membrane fusion protein of baboon reovirus is unique among the FAST proteins in that it contains two hydrophobic regions (H1 and H2) recognized as potential transmembrane (TM) domains, suggesting a polytopic topology. However, detailed topological analysis of p15 indicated only the H1 domain is membrane spanning. In the absence of an N-terminal signal peptide, the H1 TM domain serves as a reverse signal-anchor to direct p15 membrane insertion and a bitopic N(exoplasmic)/C(cytoplasmic) topology. This topology results in the translocation of the smallest ectodomain ( approximately 20 residues) of any known viral fusion protein, with the majority of p15 positioned on the cytosolic side of the membrane. Mutagenic analysis indicated the unusual presence of an N-terminal myristic acid on the small p15 ectodomain is essential to the fusion process. Furthermore, the only other hydrophobic region (H2) present in p15, aside from the TM domain, is located within the endodomain. Consequently, the p15 ectodomain is devoid of a fusion peptide motif, a hallmark feature of membrane fusion proteins. The exceedingly small, myristoylated ectodomain and the unusual topological distribution of structural motifs in this nonenveloped virus membrane fusion protein necessitate alternate models of protein-mediated membrane fusion.     

A Compact,Multifunctional Fusion Module Directs Cholesterol-Dependent Homomultimerization and Syncytiogenic Efficiency of Reovirus p10 FAST Proteins

The homologous p10 fusion-associated small transmembrane (FAST) proteins of the avian (ARV) and Nelson Bay (NBV) reoviruses are the smallest known viral membrane fusion proteins, and are virulence determinants of the fusogenic reoviruses. The small size of FAST proteins is incompatible with the paradigmatic membrane fusion pathway proposed for enveloped viral fusion proteins. Understanding how these diminutive viral fusogens mediate the complex process of membrane fusion is therefore of considerable interest, from both the pathogenesis and mechanism-of-action perspectives. Using chimeric ARV/NBV p10 constructs, the 36–40-residue ectodomain was identified as the major determinant of the differing fusion efficiencies of these homologous p10 proteins. Extensive mutagenic analysis determined the ectodomain comprises two distinct, essential functional motifs. Syncytiogenesis assays, thiol-specific surface biotinylation, and liposome lipid mixing assays identified an ∼25-residue, N-terminal motif that dictates formation of a cystine loop fusion peptide in both ARV and NBV p10. Surface immunofluorescence staining, FRET analysis and cholesterol depletion/repletion studies determined the cystine loop motif is connected through a two-residue linker to a 13-residue membrane-proximal ectodomain region (MPER). The MPER constitutes a second, independent motif governing reversible, cholesterol-dependent assembly of p10 multimers in the plasma membrane. Results further indicate that: (1) ARV and NBV homomultimers segregate to distinct, cholesterol-dependent microdomains in the plasma membrane; (2) p10 homomultimerization and cholesterol-dependent microdomain localization are co-dependent; and (3) the four juxtamembrane MPER residues present in the multimerization motif dictate species-specific microdomain association and homomultimerization. The p10 ectodomain therefore constitutes a remarkably compact, multifunctional fusion module that directs syncytiogenic efficiency and species-specific assembly of p10 homomultimers into cholesterol-dependent fusion platforms in the plasma membrane.     

The p10 FAST protein fusion peptide functions as a cystine noose to induce cholesterol-dependent liposome fusion without liposome tubulation

The reovirus p10 fusion-associated small transmembrane (FAST) proteins are the smallest known membrane fusion proteins, and evolved specifically to mediate cell–cell, rather than virus–cell, membrane fusion. The 36–40-residue ectodomains of avian reovirus (ARV) and Nelson Bay reovirus (NBV) p10 contain an essential intramolecular disulfide bond required for both cell–cell fusion and lipid mixing between liposomes. To more clearly define the functional, biochemical and biophysical features of this novel fusion peptide, synthetic peptides representing the p10 ectodomains of ARV and NBV were analyzed by solution-state NMR spectroscopy, circular dichroism spectroscopy, fluorescence spectroscopy-based hydrophobicity analysis, and liposome binding and fusion assays. Results indicate that disulfide bond formation promotes exposure of hydrophobic residues, as indicated by bis-ANS binding and time-dependent peptide aggregation under aqueous conditions, implying the disulfide bond creates a small, geometrically constrained, cystine noose. Noose formation is required for peptide partitioning into liposome membranes and liposome lipid mixing, and electron microscopy revealed that liposome–liposome fusion occurs in the absence of liposome tubulation. In addition, p10 fusion peptide activity, but not membrane partitioning, is dependent on membrane cholesterol.     

Sorting of yeast alpha 1,3 mannosyltransferase is mediated by a lumenal domain interaction, and a transmembrane domain signal that can confer clathrin-dependent Golgi localization to a secreted protein.

alpha 1,3 mannosyltransferase (Mnn1p) is a type II integral membrane protein that is localized to the yeast Golgi complex. We have examined the signals within Mnn1p that mediate Golgi localization by expression of fusion proteins comprised of Mnn1p and the secreted protein invertase. The N-terminal transmembrane domain (TMD) of Mnn1p is sufficient to localize invertase to the Golgi complex by a mechanism that is not saturable by approximately 15-20 fold overexpression. Furthermore, the TMD-mediated localization mechanism is clathrin dependent, as an invertase fusion protein bearing only the Mnn1p TMD is mislocalized to the plasma membrane of a clathrin heavy chain mutant. The Mnn1-invertase fusion proteins are not retained in the Golgi complex as efficiently as Mnn1p, suggesting that other signals may be present in the wild-type protein. Indeed, the Mnn1p lumenal domain (Mnn1-s) is also localized to the Golgi complex when expressed as a functional, soluble protein by exchanging its TMD for a cleavable signal sequence. In contrast to the Mnn1-invertase fusion proteins, overexpression of Mnn1-s saturates its retention mechanism, and results in the partial secretion of this protein. These data indicate that Mnn1p has separable Golgi localization signals within both its transmembrane and lumenal domains.     

The p97 ATPase associates with EEA1 to regulate the size of early endosomes

The AAA (ATPase-associated with various cellular activities) ATPase p97 acts on diverse substrate proteins to partake in various cellular processes such as membrane fusion and endoplasmic reticulum-associated degradation (ERAD). In membrane fusion, p97 is thought to function in analogy to the related ATPase NSF (N-ethylmaleimide-sensitive fusion protein), which promotes membrane fusion by disassembling a SNARE complex. In ERAD, p97 dislocates misfolded proteins from the ER membrane to facilitate their turnover by the proteasome. Here, we identify a novel function of p97 in endocytic trafficking by establishing the early endosomal autoantigen 1 (EEA1) as a new p97 substrate. We demonstrate that a fraction of p97 is localized to the early endosome membrane, where it binds EEA1 via the N-terminal C2H2 zinc finger domain. Inhibition of p97 either by siRNA or a pharmacological inhibitor results in clustering and enlargement of early endosomes, which is associated with an altered trafficking pattern for an endocytic cargo. Mechanistically, we show that p97 inhibition causes increased EEA1 self-association at the endosome membrane. We propose that p97 may regulate the size of early endosomes by governing the oligomeric state of EEA1.     

Biophysical and functional assays for viral membrane fusion peptides

Membrane fusion is a protein catalyzed biophysical reaction that involves the simultaneous intermixing of two phospholipid bilayers and of the aqueous compartments bound by their respective bilayers. In the case of enveloped virus fusogens, short hydrophobic or amphipathic fusion peptides that are components of the larger fusion complex are essential for the membrane merger event. The process of cell–cell membrane fusion and syncytium formation induced by the nonenveloped fusogenic orthoreoviruses is driven by the Fusion-Associated Small Transmembrane (FAST) proteins, which are similarly dependent on the action of fusion peptides. In this article, we describe some simple methods for the biophysical characterization of viral membrane fusion peptides. Liposomes serve as an ideal model system for characterizing peptide–membrane interactions because their size, shape and composition can be readily manipulated. We present details of fluorescence assays used to elucidate the kinetics of membrane fusion as well as complimentary assays used to characterize peptide-induced liposome binding and aggregation.     

In vitro fusion catalyzed by the sporulation-specific t-SNARE light-chain Spo20p is stimulated by phosphatidic acid

Sec9p and Spo20p are two SNAP25 family SNARE proteins specialized for different developmental stages in yeast. Sec9p interacts with Sso1/2p and Snc1/2p to mediate intracellular trafficking between post-Golgi vesicles and the plasma membrane during vegetative growth. Spo20p replaces Sec9p in the generation of prospore membranes during sporulation. The function of Spo20p requires enzymatically active Spo14p, which is a phosphatidylcholine (PC)-specific phospholipase D that hydrolyzes PC to generate phosphatidic acid (PA). Phosphatidic acid is required to localize Spo20p properly during sporulation; however, it seems to have additional roles that are not fully understood. Here we compared the fusion mediated by all combinations of the Sec9p or Spo20p C-terminal domains with Sso1p/Sso2p and Snc1p/Snc2p. Our results show that Spo20p forms a less efficient SNARE complex than Sec9p. The combination of Sso2p/Spo20c is the least fusogenic t-SNARE complex. Incorporation of PA in the lipid bilayer stimulates SNARE-mediated membrane fusion by all t-SNARE complexes, likely by decreasing the energetic barrier during membrane merger. This effect may allow the weak SNARE complex containing Spo20p to function during sporulation. In addition, PA can directly interact with the juxtamembrane region of Sso1p, which contributes to the stimulatory effects of PA on membrane fusion. Our results suggest that the fusion strength of SNAREs, the composition of organelle lipids and lipid-SNARE interactions may be coordinately regulated to control the rate and specificity of membrane fusion.     

Reovirus FAST Proteins Drive Pore Formation and Syncytiogenesis Using a Novel Helix-Loop-Helix Fusion-Inducing Lipid Packing Sensor

Pore formation is the most energy-demanding step during virus-induced membrane fusion, where high curvature of the fusion pore rim increases the spacing between lipid headgroups, exposing the hydrophobic interior of the membrane to water. How protein fusogens breach this thermodynamic barrier to pore formation is unclear. We identified a novel fusion-inducing lipid packing sensor (FLiPS) in the cytosolic endodomain of the baboon reovirus p15 fusion-associated small transmembrane (FAST) protein that is essential for pore formation during cell-cell fusion and syncytiogenesis. NMR spectroscopy and mutational studies indicate the dependence of this FLiPS on a hydrophobic helix-loop-helix structure. Biochemical and biophysical assays reveal the p15 FLiPS preferentially partitions into membranes with high positive curvature, and this partitioning is impeded by bis-ANS, a small molecule that inserts into hydrophobic defects in membranes. Most notably, the p15 FLiPS can be functionally replaced by heterologous amphipathic lipid packing sensors (ALPS) but not by other membrane-interactive amphipathic helices. Furthermore, a previously unrecognized amphipathic helix in the cytosolic domain of the reptilian reovirus p14 FAST protein can functionally replace the p15 FLiPS, and is itself replaceable by a heterologous ALPS motif. Anchored near the cytoplasmic leaflet by the FAST protein transmembrane domain, the FLiPS is perfectly positioned to insert into hydrophobic defects that begin to appear in the highly curved rim of nascent fusion pores, thereby lowering the energy barrier to stable pore formation.     

The pre-vacuolar t-SNARE AtPEP12p forms a 20S complex that dissociates in the presence of ATP

Many proteins are transported to the plant vacuole through the secretory pathway in small transport vesicles by a series of vesicle budding and fusion reactions. Vesicles carrying vacuolar cargo bud from the trans-Golgi network are thought to fuse with a pre-vacuolar compartment before being finally transported to the vacuole. In mammals and yeast, the fusion of a vesicle with its target organelle is mediated by a 20S protein complex containing membrane and soluble proteins that appear to be conserved between different species. A number of membrane proteins have been identified in plants that show sequence similarity with a family of integral membrane proteins (t-SNAREs) on target organelles that are required for the fusion of transport vesicles with that organelle. However, the biochemical function of these proteins has remained elusive. Here, we demonstrate for the first time the formation of a 20S complex in plants that has characteristics of complexes involved in vesicle fusion. This complex contains AtPEP12p, an Arabidopsis protein thought to be involved in protein transport to the prevacuolar compartment. In addition, we have shown that AtPEP12p can bind to alpha-SNAP, indicating that AtPEP12p does indeed function as a SNAP receptor or SNARE. These preliminary data suggest that AtPEP12p may function jointly with alpha-SNAP and NSF in the fusion of transport vesicles containing vacuolar cargo proteins with the pre-vacuolar compartment.     

Different import pathways through the mitochondrial intermembrane space for inner membrane proteins.

Earlier work on the protein import system of yeast mitochondria has identified two soluble 70 kDa protein complexes in the intermembrane space. One complex contains the essential proteins Tim9p and Tim10p and mediates transport of cytosolically-made metabolite carrier proteins from the outer to the inner membrane. The other complex contains the non-essential proteins Tim8p and Tim13p as well as loosely associated Tim9p; its function was unclear, but it interacted structurally or functionally with the Tim9p-Tim10p complex. We now show that the two 70 kDa complexes each mediate the import of a different subset of integral inner membrane proteins and that they can transfer these proteins to one of three different membrane insertion sites: the TIM22 complex, the TIM23 complex or an as yet uncharacterized insertion site. Yeast mitochondria thus use multiple pathways for escorting hydrophobic inner membrane proteins across the aqueous intermembrane space.