Got1p and Sft2p: membrane proteins involved in traffic to the Golgi complex.
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Traffic through the yeast Golgi complex depends on a member of the syntaxin family of SNARE proteins, Sed5p, present in early Golgi cisternae. Sft2p is a non-essential tetra-spanning membrane protein, found mostly in the late Golgi, that can suppress some sed5 alleles. We screened for mutations that show synthetic lethality with sft2 and found one that affects a previously uncharacterized membrane protein, Got1p, as well as new alleles of sed5 and vps3. Got1p is an evolutionarily conserved non-essential protein with a membrane topology similar to that of Sft2p. Immunofluorescence and subcellular fractionation indicate that it is present in early Golgi cisternae. got1 mutants, but not sft2 mutants, show a defect in an in vitro assay for ER-Golgi transport at a step after vesicle tethering to Golgi membranes. In vivo, inactivation of both Got1p and Sft2p results in phenotypes ascribable to a defect in endosome-Golgi traffic, while their complete removal results in an ER-Golgi transport defect. Thus the presence of either Got1p or Sft2p is required for vesicle fusion with the Golgi complex in vivo. We suggest that Got1p normally facilitates Sed5p-dependent fusion events, while Sft2p performs a related function in the late Golgi. (+info)
New insights into an old protein: the functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase.
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Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was considered a classical glycolytic protein examined for its pivotal role in energy production. It was also used as a model protein for analysis of protein structure and enzyme mechanisms. The GAPDH gene was utilized as a prototype for studies of genetic organization, expression and regulation. However, recent evidence demonstrates that mammalian GAPDH displays a number of diverse activities unrelated to its glycolytic function. These include its role in membrane fusion, microtubule bundling, phosphotransferase activity, nuclear RNA export, DNA replication and DNA repair. These new activities may be related to the subcellular localization and oligomeric structure of GAPDH in vivo. Furthermore, other investigations suggest that GAPDH is involved in apoptosis, age-related neurodegenerative disease, prostate cancer and viral pathogenesis. Intriguingly, GAPDH is also a unique target of nitric oxide. This review discusses the functional diversity of GAPDH in relation to its protein structure. The mechanisms through which mammalian cells may utilize GAPDH amino acid sequences to provide these new functions and to determine its intracellular localization are considered. The interrelationship between new GAPDH activities and its role in cell pathologies is addressed. (+info)
Relationship of membrane sidedness to the effects of the lipophosphoglycan of Leishmania donovani on the fusion of influenza virus.
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Cells expressing the influenza hemagglutinin protein were fused to planar lipid bilayers containing the viral receptor GD1a at pH 5.0. An amphiphile known to alter membrane properties is lipophosphoglycan (LPG). This glycoconjugate was added from aqueous solution to either the cis or the trans monolayer to examine its effects on the fusion process. LPG markedly inhibited the formation of fusion pores when present in the cis monolayer but LPG in the trans monolayer had no effect on the parameters of pore formation or on the properties of the pores. The N-terminal segment of the HA2 subunit of the influenza hemagglutinin protein is important for membrane fusion. The effect of LPG on the conformation and membrane insertion of a synthetic 20-amino-acid peptide, corresponding to the influenza fusion peptide, was examined at pH 5.0 by attenuated total reflection Fourier transform infrared spectroscopy and by the fluorescence properties of the Trp residues of this peptide. It was found that cis LPG did not prevent insertion of the peptide into the membrane but it did alter the conformation of the membrane-inserted peptide from alpha-helix to beta-structure. The beta-structure was oriented along the bilayer normal. The effect of cis LPG on the conformation of the fusion peptide probably contributes to the observed inhibition of pore formation and lipid mixing. In contrast, trans LPG has no effect on the conformation or angle of membrane insertion of the peptide, nor does it affect pore formation by HA-expressing cells. The ineffectiveness of trans LPG, despite it having strong positive curvature-promoting properties, may be a consequence of the size of this amphiphile being too large to enter a fusion pore. (+info)
Acidic pH enhancement of the fusion of Newcastle disease virus with cultured cells.
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Fusion of the lentogenic strain "Clone 30" of Newcastle disease virus (NDV) with the cell line COS-7 has been studied. Fusion was monitored using the octadecylrhodamine B chloride dequenching assay [Hoekstra, D., de Boer, T., Klappe, K. and Wilschut, J. (1984). Biochemistry 23, 5675-5681]. In the present work, fusion of NDV with COS-7 cells was found to occur in a time- and temperature-dependent fashion. Significant dequenching of the probe occurred at temperatures higher than 28 degrees C. A 20-fold excess of unlabeled virus inhibited fusion by about 53% compared with the control, whereas 62% inhibition of fusion was obtained after digestion of viral glycoproteins with trypsin. The data are discussed in terms of the nonfusion transfer of the probe. In addition, preincubation of cells with 50 mM ammonium chloride or 0.1% sodium azide prevented NDV from fusing with COS-7 cells by about 30% in comparison with the control. The cytopathic effect of NDV infection in cell culture in the presence of ammonium chloride was reduced compared with control. Moreover, viral preincubation at pH 5 yielded a mild inhibition of fusogenic activity. Our results suggest that NDV may use the endocytic pathway as a complementary way of entering cells by direct fusion with the plasma membrane. (+info)
Stimulation of glycosylphosphatidylinositol biosynthesis in mammalian cell-free systems by GTP hydrolysis: evidence for the involvement of membrane fusion.
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The second step in glycosylphosphatidylinositol (GPI) biosynthesis, the deacetylation of GlcNAc-phosphatidylinositol (GlcNAc-PI), has been shown to be stimulated by GTP hydrolysis [Stevens (1993) J. Biol. Chem. 268, 9718-9724]. We have now developed a system to study this regulation that uses microsomes from cells defective in the first step in GPI biosynthesis (class A, C and H lymphoma mutants) and the second reaction in the pathway (G9PLAP.85). With this mixed-microsome system, the deacetylation of GlcNAc-PI was almost completely dependent on GTP hydrolysis. Because GlcNAc-PI synthesized by the G9PLAP.85 microsomes cannot readily move to the first-step-mutant microsomes to be deacetylated, this result indicated that the role of GTP was to facilitate the 'apparent' transfer of this substrate between membrane vesicles. The microsomes could be stably preactivated by pretreatment with GTP before GPI biosynthesis was initiated, indicating that fusion was the most likely mechanism for this regulation. GlcNAc-PI deacetylation could also be stably preactivated in EL4 microsomes, suggesting that fusion also occurred in wild-type membranes. Some differential localization of the GlcNAc-PI synthetic and deacetylation activities with the endoplasmic reticulum was found. Therefore fusion seems to stimulate GPI biosynthesis in mammalian microsomes by bringing together the first two enzymes in the pathway in the same membrane vesicle. (+info)
Depletion of syntaxins in the early Caenorhabditis elegans embryo reveals a role for membrane fusion events in cytokinesis.
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BACKGROUND: During cytokinesis, the plasma membrane of the parent cell is resolved into the two plasma membranes of the daughter cells. Membrane fusion events mediated by the machinery that participates in intracellular vesicle trafficking might contribute to this process. Two classes of molecules that are required for membrane fusion are the t-SNAREs and the v-SNAREs. The t-SNAREs (syntaxins) comprise a multi-gene family that has been suggested to mediate, at least in part, selective membrane fusion events in the cell. RESULTS: We have analyzed the genome of Caenorhabditis elegans and identified eight syntaxin genes. RNA-mediated interference (RNAi) was used to produce embryos deficient in individual syntaxins and these embryos were phenotypically characterized. Embryos deficient in one syntaxin, Syn-4, became multinucleate because of defects in karyomere fusion and cytokinesis. Syn-4 localized both to ingressing cleavage furrows and to punctate structures surrounding nuclei as they reformed during interphase. CONCLUSIONS: Our analyses indicate that both cytokinesis and reformation of the nuclear envelope are dependent on SNARE-mediated membrane fusion. (+info)
Ultrastructural characterization of peptide-induced membrane fusion and peptide self-assembly in the lipid bilayer.
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The peptide sequence B18, derived from the membrane-associated sea urchin sperm protein bindin, triggers fusion between lipid vesicles. It exhibits many similarities to viral fusion peptides and may have a corresponding function in fertilization. The lipid-peptide and peptide-peptide interactions of B18 are investigated here at the ultrastructural level by electron microscopy and x-ray diffraction. The histidine-rich peptide is shown to self-associate into two distinctly different supramolecular structures, depending on the presence of Zn(2+), which controls its fusogenic activity. In aqueous buffer the peptide per se assembles into beta-sheet amyloid fibrils, whereas in the presence of Zn(2+) it forms smooth globular clusters. When B18 per se is added to uncharged large unilamellar vesicles, they become visibly disrupted by the fibrils, but no genuine fusion is observed. Only in the presence of Zn(2+) does the peptide induce extensive fusion of vesicles, which is evident from their dramatic increase in size. Besides these morphological changes, we observed distinct fibrillar and particulate structures in the bilayer, which are attributed to B18 in either of its two self-assembled forms. We conclude that membrane fusion involves an alpha-helical peptide conformation, which can oligomerize further in the membrane. The role of Zn(2+) is to promote this local helical structure in B18 and to prevent its inactivation as beta-sheet fibrils. (+info)
Tension of membranes expressing the hemagglutinin of influenza virus inhibits fusion.
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The effects of membrane tension on fusion between cells expressing the hemagglutinin (HA) of influenza virus and red blood cells were studied by capacitance measurements. Inflation of an HA-expressing cell was achieved by applying a positive hydrostatic pressure to its interior through a patch-clamp pipette in the whole-cell configuration. Inflating cells to the maximum extent possible without lysis created a membrane tension and completely inhibited low-pH-induced fusion at room temperature. Fully inflated cells that were subsequently deflated to normal size resumed the ability to fuse in response to low pH. At the higher temperature of 32 degrees C, fusion conditions were sufficiently optimal that full inflation did not hinder fusion, and once formed, pores enlarged more rapidly than those of never inflated cells. It is suggested that under fusogenic conditions HA causes the formation of a dimple within the membrane in which it resides, and that membrane tension hinders fusion by preventing the formation of dimples. Because dimpling bends the bilayer portion of bound membranes so that they come into intimate contact, the damping of dimpling would suppress this initial step in the fusion process. (+info)