Membrane Fusion Function Research Articles

Calcium-dependent regulation of exocytosis

A rapid increase in intracellular calcium directly triggers regulated exocytosis. In addition, changes in intracellular calcium concentration can adjust the extent of exocytosis (quantal content) or the magnitude of individual release events (quantal size) in both the short- and long-term. It is generally agreed that calcium achieves this regulation via an interaction with a number of different molecular targets located at or near to the site of membrane fusion. We review here the synaptic proteins with defined calcium-binding domains and protein kinases activated by calcium, summarize what is known about their function in membrane fusion and the experimental evidence in support of their involvement in synaptic plasticity.

The v-ATPase V0 Subunit a1 Is Required for a Late Step in Synaptic Vesicle Exocytosis in Drosophila

The V(0) complex forms the proteolipid pore of an ATPase that acidifies vesicles. In addition, an independent function in membrane fusion has been proposed largely based on yeast vacuolar fusion experiments. We have isolated mutations in the largest V(0) component vha100-1 in flies in an unbiased genetic screen for synaptic malfunction. The protein is only required in neurons, colocalizes with markers for synaptic vesicles as well as active zones, and interacts with t-SNAREs. Loss of vha100-1 leads to vesicle accumulation in synaptic terminals, suggesting a deficit in release. The amplitude of spontaneous release events and release with hypertonic stimulation indicate normal levels of neurotransmitter loading, yet mutant embryos display severe defects in evoked synaptic transmission and FM1-43 uptake. Our data suggest that Vha100-1 functions downstream of SNAREs in synaptic vesicle fusion.

A Salmonella typhimurium Effector Protein SifA Is Modified by Host Cell Prenylation and S-Acylation Machinery

SifA is a Salmonella effector protein that is required for maintenance of the vacuolar membrane that surrounds replicating bacteria. It associates with the Salmonella-containing vacuole but how it interacts with the membrane is unknown. Here we show by immunofluorescence, S100 fractionation and Triton X-114 partitioning that the membrane association and targeting properties of SifA are influenced by a motif encoded within the C-terminal six amino acids. This sequence shares homology with both CAAX and Rab geranylgeranyl transferase prenylation motifs. We characterized the post-translational processing of SifA and showed that the cysteine residue within the CAAX motif is modified by isoprenoid addition through the action of protein geranylgeranyl transferase I. SifA was additionally modified by S-acylation of an adjacent cysteine residue. Similar modifications to host cell proteins regulate numerous functions including protein targeting, membrane association, protein-protein interaction, and signal transduction. This is the only known example of a bacterial effector protein that is modified both by mammalian cell S-acylation and prenylation machinery.

New nuclear functions of the glycolytic protein, glyceraldehyde‐3‐phosphate dehydrogenase, in mammalian cells

Recent studies establish that the glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is not simply a classical metabolic protein involved in energy production. Instead, it is a multifunctional protein with defined functions in numerous subcellular processes. New investigations establish a primary role for GAPDH in a variety of critical nuclear pathways apart from its already recognized role in apoptosis. These new roles include its requirement for transcriptional control of histone gene expression, its essential function in nuclear membrane fusion, its necessity for the recognition of fraudulently incorporated nucleotides in DNA, and its mandatory participation in the maintenance of telomere structure. Each of these new functions requires GAPDH association into a series of multienzyme complexes. Although other proteins in those complexes are variable, GAPDH remains the single constant protein in each structure. To undertake these new functions, GAPDH is recruited to the nucleus in S phase or its intracellular distribution is regulated as a function of drug exposure. Other investigations relate a substantial role for nuclear GAPDH in hyperglycemic stress and the development of metabolic syndrome. Considerations of future directions as well as the role of GAPDH post-translational modification as a basis for its multifunctional activities is suggested.

The Conserved Glycine-Rich Segment Linking the N-Terminal Fusion Peptide to the Coiled Coil of Human T-Cell Leukemia Virus Type 1 Transmembrane Glycoprotein gp21 Is a Determinant of Membrane Fusion Function

Retroviral transmembrane proteins (TMs) contain an N-terminal fusion peptide that initiates virus-cell membrane fusion. The fusion peptide is linked to the coiled-coil core through a conserved sequence that is often rich in glycines. We investigated the functional role of the glycine-rich segment, Met-326 to Ser-337, of the human T-cell leukemia virus type 1 (HTLV-1) TM, gp21, by alanine and proline scanning mutagenesis. Alanine substitution for the hydrophobic residue Ile-334 caused an approximately 90% reduction in cell-cell fusion activity without detectable effects on the lipid-mixing and pore formation phases of fusion. Alanine substitutions at other positions had smaller effects (Gly-329, Val-330, and Gly-332) or no effect on fusion function. Proline substitution for glycine residues inhibited cell-cell fusion function with position-dependent effects on the three phases of fusion. Retroviral glycoprotein fusion function thus appears to require flexibility within the glycine-rich segment and hydrophobic contacts mediated by this segment.

The Fusion-Controlling Disulfide Bond Isomerase in Retrovirus Env Is Triggered by Protein Destabilization

The membrane fusion function of murine leukemia virus (MLV) is carried by the Env protein. This protein is composed of three SU-TM subunit complexes. The fusion activity is loaded into the transmembrane TM subunit and controlled by the peripheral, receptor-binding SU subunit. It is assumed that TM adopts a metastable conformation in the native Env and that fusion activation involves the folding of TM into a stable form. Activation is suppressed by the associated SU and triggered by its dissociation, which follows receptor binding. Recently we showed that the two subunits are disulfide linked and that SU dissociation and triggering of the fusion function are caused by a switch of the intersubunit disulfide into an intrasubunit disulfide isomer using an isomerization-active CWLC motif in SU (M. Wallin, M. Ekstrom, and H. Garoff, EMBO J. 23:54-65, 2004). In the present work we address how the SU disulfide isomerase is activated. Using Moloney MLV, we show that isomerization of the SU-TM disulfide bond can be triggered by heat, urea, or guanidinium hydrochloride. Such protein perturbation treatments also significantly increase the kinetics and efficiency of viral fusion. The threshold conditions for the effects on isomerization and fusion are virtually the same. This finding indicates that destabilization of interactions in the SU oligomer induces the disulfide bond isomerase and the subsequent activation of the fusion function in TM.

Budding of alphaviruses

Alphaviruses are small highly ordered enveloped RNA viruses, which replicate very efficiently in the infected cell. They consist of a nucleocapsid (NC) and a surrounding membrane with glycoproteins. In the NC the positive single stranded RNA genome of the virus is enclosed by a T = 4 icosahedral shell of capsid (C) proteins. The glycoproteins form a second shell with corresponding symmetry on the outside of the lipid membrane. These viruses mature by budding at the plasma membrane (PM) of the infected cell and enter into new cells by acid-triggered membrane fusion in endosomes. The viral glycoprotein consists of two subunits, E1, which carries the membrane fusion function, and E2, which suppresses this function until acid activation occurs. In the infected cell the RNA replication and transcription are confined to the cytoplasmic surface of endosome-derived vesicles called cytopathic vacuoles type I (CPV I). These structures are closely associated with membranes of the endoplasmic reticulum (ER), thereby creating a microenvironment for synthesis of viral proteins, assembly of the glycoproteins and formation of genome-C complexes. The budding process of the virus is initiated by C-glycoprotein interactions, possibly already before the glycoproteins arrive at the PM. This might involve a premade, ordered NC or a less ordered form of the genome-C complex. In the latter case, the interactions in the glycoprotein shell provide the major driving force for budding. The nature of the C-glycoprotein interaction has been resolved at atomic resolution by modelling. It involves hydrophobic interactions between a Tyr–X–Leu tripeptide in the internal tail of the E2 subunit and a pocket on the surface of the C protein. When the virus enters the endosome of a new cell the acid conditions trigger rearrangements in the glycoprotein shell, which result in the dissociation of the interactions that drive budding and a concomitant activation of the membrane fusion function in the E1 subunit.

732. A Novel Double-Pseudotyped Viral Vector for Gene Therapy

A major goal of gene therapy is to achieve highly efficient and specific delivery of therapeutic genes into target cells. While current gene delivery vectors have been successful in many cases, there are still some barriers. These include low titer, low specificity, short-term expression, inconvenient vector design for different targets and inefficient targeting of certain tissues due to the lack of known receptors. For retroviral-based vectors, one limitation is the low flexibility in modifying the viral envelope glycoprotein to achieve the desired tropism since the receptor binding domain (SU) and the fusion domain (TM) are coupled. In an attempt to improve some of these aspects, we have designed a novel two-molecule targeting system that employs a lentiviral-based double-pseudotyped vector in which two different proteins are incorporated into the viral membrane to separate the receptor binding and membrane fusion functions. The first molecule, a mutant form of influenza hemagglutinin (HA) deficient for intrinsic receptor-binding function, supplies the membrane fusion machinery. The second molecule, a modified cellular surface protein that encodes two copies of the IgG-binding domain from staphylococcal protein A, binds to specific receptors on target cells through antibodies used to coat the virus. Specificity of the vector can be readily altered by employing a different antibody.

The baculovirus GP64 protein mediates highly stable infectivity of a human respiratory syncytial virus lacking its homologous transmembrane glycoproteins.

Baculovirus GP64 is a low-pH-dependent membrane fusion protein required for virus entry and cell-to-cell transmission. Recently, GP64 has generated interest for practical applications in mammalian systems. Here we examined the membrane fusion function of GP64 from Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressed in mammalian cells, as well as its capacity to functionally complement a mammalian virus, human respiratory syncytial virus (HRSV). Both authentic GP64 and GP(64/F), a chimeric protein in which the GP64 cytoplasmic tail domain was replaced with the 12 C-terminal amino acids of the HRSV fusion (F) protein, induced low-pH-dependent cell-cell fusion when expressed transiently in HEp-2 (human) cells. Levels of surface expression and syncytium formation were substantially higher at 33 degrees C than at 37 degrees C. The open reading frames (ORFs) encoding GP64 or GP(64/F), along with two marker ORFs encoding green fluorescent protein (GFP) and beta-glucuronidase (GUS), were used to replace all three homologous transmembrane glycoprotein ORFs (small hydrophobic SH, attachment G, and F) in a cDNA of HRSV. Infectious viruses were recovered that lacked the HRSV SH, G, and F proteins and expressed instead the GP64 or GP(64/F) protein and the two marker proteins GFP and GUS. The properties of these viruses, designated RSDeltaSH,G,F/GP64 or RSDeltaSH,G,F/GP(64/F), respectively, were compared to a previously described HRSV expressing GFP in place of SH but still containing the wild-type HRSV G and F proteins (RSDeltaSH [A. G. Oomens, A. G. Megaw, and G. W. Wertz, J. Virol., 77:3785-3798, 2003]). By immunoelectron microscopy, the GP64 and GP(64/F) proteins were shown to incorporate into HRSV-induced filaments at the cell surface. Antibody neutralization, ammonium chloride inhibition, and replication levels in cell culture showed that both GP64 proteins efficiently mediated infectivity of the respective viruses in a temperature-sensitive, low-pH-dependent manner. Furthermore, RSDeltaSH,G,F/GP64 and RSDeltaSH,G,F/GP(64/F) replicated to higher levels and had significantly higher stability of infectivity than HRSVs containing the homologous HRSV G and F proteins. Thus, GP64 and a GP64/HRSV F chimeric protein were functional and efficiently complemented an unrelated human virus in mammalian cells, producing stable, infectious virus stocks. These results demonstrate the potential of GP64 for both practical applications requiring stable pseudotypes in mammalian systems and for studies of viral glycoprotein requirements in assembly and pathogenesis.

Functional Evolution of the HIV-1 Envelope Glycoprotein 120 Association Site of Glycoprotein 41

Protein-protein interaction surfaces can exhibit structural plasticity, a mechanism whereby an interface adapts to mutations as binding partners coevolve. The HIV-1 envelope glycoprotein gp120-gp41 complex, which is responsible for receptor attachment and membrane fusion, represents an extreme example of a coevolving complex as up to 35% amino acid sequence divergence has been observed in these proteins among HIV-1 isolates. In this study, the function of conserved gp120 contact residues, Leu593, Trp596, Gly597, Lys601, and Trp610 within the disulfide-bonded region of gp41, was examined in envelope glycoproteins derived from diverse HIV-1 isolates. We found that the gp120-gp41 association function of the disulfide-bonded region is conserved. However, the contribution of individual residues to gp41 folding and/or stability, gp120-gp41 association, membrane fusion function, and viral entry varied from isolate to isolate. In gp120-gp41 derived from the dual-tropic isolate, HIV-189.6, the importance of Trp596 for fusion function was dependent on the chemokine receptor utilized as a fusion cofactor. Thus, the engagement of alternative chemokine receptors may evoke distinct fusion-activation signals involving the site of gp120-gp41 association. An examination of chimeric glycoproteins revealed that the isolate-specific functional contributions of particular gp120-contact residues are influenced by the sequence of gp120 hypervariable regions 1, 2, and 3. These data indicate that the gp120-gp41 association site is structurally and functionally adaptable, perhaps to maintain a functional glycoprotein complex in a setting of host selective pressures driving the rapid coevolution of gp120 and gp41.

Cdc42p functions at the docking stage of yeast vacuole membrane fusion.

Membrane fusion reactions have been considered to be primarily regulated by Rab GTPases. In the model system of homotypic vacuole fusion in the yeast Saccharomyces cerevisiae, we show that Cdc42p, a member of the Rho family of GTPases, has a direct role in membrane fusion. Genetic evidence suggested a relationship between Cdc42p and Vtc1p/Nrf1p, a central part of the vacuolar membrane fusion machinery. Vacuoles from cdc42 temperature-sensitive mutants are deficient for fusion at the restrictive temperature. Specific amino acid changes on the Cdc42p protein surface in these mutants define the putative interaction domain that is crucial for its function in membrane fusion. Affinity-purified antibodies to this domain inhibited the in vitro fusion reaction. Using these antibodies in kinetic analyses and assays for subreactions of the priming, docking and post-docking phase of the reaction, we show that Cdc42p action follows Ypt7p-dependent tethering, but precedes the formation of trans-SNARE complexes. Thus, our data define an effector binding domain of Cdc42p by which it regulates the docking reaction of vacuole fusion.

A putative endosomal t-SNARE links exo- and endocytosis in the phytopathogenic fungus Ustilago maydis.

We identified a temperature-sensitive mutant of the plant pathogenic fungus Ustilago maydis that is defective in the polar distribution of cell wall components and shows abnormal morphology. The affected gene, yup1, was cloned by complementation. It encodes a putative target soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (t-SNARE), suggesting a function in membrane fusion. A Yup1-GFP fusion protein localized to vesicles that showed rapid saltatory motion along microtubules. These vesicles are part of the endocytic pathway and accumulate at sites of active growth, thereby supporting the expansion of the hyphal tip. In yup1(ts) cells, endocytosis is impaired and accumulation of Yup1-carrying endosomes at cell poles is abolished, resulting in apolar distribution of wall components and morphological alterations. This suggests that a membrane recycling process via early endosomes supports polar growth of U. maydis.

Antigen-specific membrane fusion mediated by the haemagglutinin protein of influenza A virus: separation of attachment and fusion functions on different molecules.

Using genetic engineering techniques, two strategies for changing the receptor-binding specificity of the influenza A virus haemagglutinin (HA) protein whilst preserving its membrane fusion function, have been explored. The aim was to investigate whether the HA protein could be developed as an attachment/entry protein for targeting enveloped virus gene therapy vectors to specific cell populations. In the first strategy, a single chain antibody Fv region (scFv) specific for the hapten NIP was inserted between HA1 codons 139 and 145, to create a scFv-HA chimaeric protein. This protein was shown to possess anti-NIP binding activity, but membrane fusion activity could not be demonstrated. The possibility that linking the scFv domain directly to HA may have inhibited the HA fusion function led to the development of the second strategy. This involved separating the receptor-binding and membrane fusion functions of HA on to two different molecules. The feasibility of this strategy was tested by looking for fusion between NIP-conjugated red blood cells which lacked sialic acid (the HA protein's natural receptor) and Chinese hamster ovary cells that expressed both the above anti-NIP scFv-HA chimaeric protein (as a non-fusigenic, receptor-binding molecule) and wild-type HA protein (as a fusigenic, non-binding molecule) on their surface. Cell-to-cell fusion was observed in this system, indicating that the receptor-binding function of HA can be transferred to an adjacent molecule, and also changed in its specificity, without compromising its membrane fusion activity. This finding strongly suggests that the development of a two-molecule attachment and entry system for retargeting enveloped virus gene therapy vectors, based on HA, is a viable proposition.

Newly Synthesized Phosphatidylinositol Phosphates Are Required for Synaptic Norepinephrine but Not Glutamate or γ-Aminobutyric Acid (GABA) Release

Newly synthesized phosphatidylinositol phosphates have been implicated in many membrane-trafficking reactions. They are essential for exocytosis of norepinephrine in PC12 cells and chromaffin cells, suggesting a function in membrane fusion. We have now studied the role of phosphatidylinositol phosphates in synaptic vesicle exocytosis using synaptosomes. Under conditions where phosphorylation of phosphatidylinositols is blocked, norepinephrine secretion was nearly abolished whereas glutamate and GABA release was still elicited. Thus phosphatidylinositides are essential only for some membrane fusion reactions, and exocytotic release mechanisms differ between neurotransmitters.

Characterization of a Hemagglutinin-Specific Inhibitor of Influenza A Virus

A novel inhibitor of influenza virus growth in tissue culture was identified and characterized. This compound (BMY-27709) has an IC50of 3–8 μMfor A/WSN/33 virus growth in a multicycle replication assay and is active against all H1 and H2 subtype viruses tested. However, BMY-27709 was found to be inactive against H3 subtype viruses, as well as influenza B/Lee/40 virus. BMY-27709 was also found to act against H1 and H2 viruses early in infection, suggesting that the target for inhibition is the hemagglutinin protein. This was confirmed through the use of reassortant viruses and the isolation of a virus resistant to BMY-27709. The resistant virus isolated contained a phenylalanine to serine change at amino acid 110 of the HA2 subunit. That this single mutation was responsible for the acquisition of resistance to BMY-27709 was proven through reverse genetics, as transfectant virus containing only this change was shown to be resistant to BMY-27709, while the control virus without this mutation remained sensitive. BMY-27709 is able to inhibit virus-induced red blood cell hemolysis, suggesting that it blocks the membrane fusion function of hemagglutinin. These experiments further illustrate that the hemagglutinin protein of influenza virus is a viable target for the discovery and development of small molecule inhibitors of virus growth.

Both the V2 and V3 regions of the human immunodeficiency virus type 1 surface glycoprotein functionally interact with other envelope regions in syncytium formation.

To map the regions of the external envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) involved in the process of membrane fusion, we determined the syncytium-inducing capacity of a panel of transiently expressed chimeric envelope genes. This panel was generated by exchanging gene fragments between four previously studied envelope genes that exhibited a high degree of sequence homology yet displayed marked differences in syncytium-inducing capacity when expressed by recombinant vaccinia virus. The results demonstrate that multiple regions of the HIV-1 envelope glycoproteins are involved in syncytium formation. Some fragments, most notably those containing the V2 or V3 region, can transfer syncytium-inducing capacity to envelope proteins previously not capable of inducing syncytia. Moreover, it is shown that such regions functionally interact with other envelope regions, especially one encompassing the V4 and V5 regions of gp120 or a region encompassing part of gp41, to exert their function in membrane fusion.

Anti-peptide antibodies detect steps in a protein conformational change: low-pH activation of the influenza virus hemagglutinin.

At low pH, the hemagglutinin (HA) of influenza virus undergoes an irreversible conformational change that potentiates its essential membrane fusion function. We have probed the details of this conformational change using a panel of 14 anti-HA-peptide antibodies. Whereas some antibodies reacted equally well with both the neutral and low-pH HA conformations, others reacted to a significantly greater extent with the low-pH form. The locations of the peptides recognized by the latter antibodies in the three-dimensional HA structure indicated regions of the protein that change in response to low pH. Moreover, kinetic experiments suggested steps in the conformational change. In addition to their relevance to membrane fusion, our results show that anti-peptide antibodies can be used to study some types of biologically important protein conformational changes.

Isolation and characterization of the measles virus F 1 polypeptide: Comparison with other paramyxovirus fusion proteins

Measles virus fusion (F) protein has been isolated by immunoadsorption to a complex of monoclonal antibodies bound to protein A-Sepharose. The 41-kDa F 1 component of the fusion protein was obtained pure in high yield by preparative SDS-polyacrylamide gel electrophoresis. The amino acid composition of the F 1 chain was determined and the N-terminal sequence was analyzed for 40 residues. The structure determined is largely hydrophobic, with 24 residues of Val, Ile, Leu, Met, Phe, or Ala. Comparison with previously published data on the F 1 polypeptide of Sendai virus showed considerable similarity in amino acid composition. Extensive N-terminal sequence homologies with F 1 polypeptides of different paramyxoviruses are also noticed, including a nine-residue segment strictly conserved among four F 1 polypeptides studied, as well as a weaker but distinct and Glyrich sequence homology with the influenza A and B virus HA 2 polypeptides. The evolutionary conservation of the N-terminal region at the site of cleavage of surface glycoproteins of the two families of myxoviruses highlights its specialized function in membrane fusion.

Isolation and characterization of the measles virus F 1 Polypeptide. Comparison with other paramyxovirus fusion proteins

Abstract Measles virus fusion (F) protein has been isolated by immunoadsorption to a complex of monoclonal antibodies bound to protein A-Sepharose. The 41-kDa F 1 component of the fusion protein was obtained pure in high yield by preparative SDS-polyacrylamide gel electrophoresis. The amino acid composition of the F 1 chain was determined and the N-terminal sequence was analyzed for 40 residues. The structure determined is largely hydrophobic, with 24 residues of Val, Ile, Leu, Met, Phe, or Ala. Comparison with previously published data on the F 1 polypeptide of Sendai virus showed considerable similarity in amino acid composition. Extensive N-terminal sequence homologies with F 1 polypeptides of different paramyxoviruses are also noticed, including a nine-residue segment strictly conserved among four F 1 polypeptides studied, as well as a weaker but distinct and Glyrich sequence homology with the influenza A and B virus HA 2 polypeptides. The evolutionary conservation of the N-terminal region at the site of cleavage of surface glycoproteins of the two families of myxoviruses highlights its specialized function in membrane fusion.