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1.
Protein Sci ; 8(11): 2465-73, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10595551

ABSTRACT

In protein transport between organelles, interactions of v- and t-SNARE proteins are required for fusion of protein-containing vesicles with appropriate target compartments. Mammalian SNARE proteins have been observed to interact with NSF and SNAP, and yeast SNAREs with yeast homologues of NSF and SNAP proteins. This observation led to the hypothesis that, despite low sequence homology, SNARE proteins are structurally similar among eukaryotes. SNARE proteins can be classified into two groups depending on whether they interact with SNARE binding partners via conserved glutamine (Q-SNAREs) or arginine (R-SNAREs). Much of the published structural data available is for SNAREs involved in exocytosis (either in yeast or synaptic vesicles). This paper describes circular dichroism, Fourier transform infrared spectroscopy, and dynamic light scattering data for a set of yeast v- and t-SNARE proteins, Vti1p and Pep12p, that are Q-SNAREs involved in intracellular trafficking. Our results suggest that the secondary structure of Vti1p is highly alpha-helical and that Vti1p forms multimers under a variety of solution conditions. In these respects, Vti1p appears to be distinct from R-SNARE proteins characterized previously. The alpha-helicity of Vti1p is similar to that of Q-SNARE proteins characterized previously. Pep12p, a Q-SNARE, is highly alpha-helical. It is distinct from other Q-SNAREs in that it forms dimers under many of the solution conditions tested in our experiments. The results presented in this paper are among the first to suggest heterogeneity in the functioning of SNARE complexes.


Subject(s)
Carrier Proteins/chemistry , Membrane Proteins/chemistry , Saccharomyces cerevisiae Proteins , Vesicular Transport Proteins , Amino Acid Sequence , Animals , Cloning, Molecular , Fungal Proteins/chemistry , Light , Mammals , Molecular Sequence Data , Protein Conformation , Protein Structure, Secondary , Qa-SNARE Proteins , Qb-SNARE Proteins , Recombinant Proteins , Saccharomyces cerevisiae/metabolism , Scattering, Radiation
2.
Mol Biol Cell ; 10(6): 1719-32, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10359592

ABSTRACT

The interaction between v-SNAREs on transport vesicles and t-SNAREs on target membranes is required for membrane traffic in eukaryotic cells. Here we identify Vti1p as the first v-SNARE protein found to be required for biosynthetic traffic into the yeast vacuole, the equivalent of the mammalian lysosome. Certain vti1-ts yeast mutants are defective in alkaline phosphatase transport from the Golgi to the vacuole and in targeting of aminopeptidase I from the cytosol to the vacuole. VTI1 interacts genetically with the vacuolar t-SNARE VAM3, which is required for transport of both alkaline phosphatase and aminopeptidase I to the vacuole. The v-SNARE Nyv1p forms a SNARE complex with Vam3p in homotypic vacuolar fusion; however, we find that Nyv1p is not required for any of the three biosynthetic pathways to the vacuole. v-SNAREs were thought to ensure specificity in membrane traffic. However, Vti1p also functions in two additional membrane traffic pathways: Vti1p interacts with the t-SNAREs Pep12p in traffic from the TGN to the prevacuolar compartment and with Sed5p in retrograde traffic to the cis-Golgi. The ability of Vti1p to mediate multiple fusion steps requires additional proteins to ensure specificity in membrane traffic.


Subject(s)
Carrier Proteins/metabolism , Fungal Proteins/metabolism , Intracellular Membranes/metabolism , Membrane Proteins/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Vacuoles/metabolism , Vesicular Transport Proteins , Alkaline Phosphatase/metabolism , Aminopeptidases/metabolism , Biological Transport , Golgi Apparatus/metabolism , Qa-SNARE Proteins , Qb-SNARE Proteins
3.
J Biol Chem ; 273(5): 2624-30, 1998 Jan 30.
Article in English | MEDLINE | ID: mdl-9446565

ABSTRACT

Membrane traffic in eukaryotic cells requires the interaction of a vesicle-associated soluble NSF attachment protein receptor (v-SNARE) on transport vesicles with a SNARE on the target membrane (t-SNARE). Recently, we identified the yeast protein Vti1p as a v-SNARE that is involved in two transport reactions. Vti1p interacts with the prevacuolar t-SNARE Pep12p in Golgi to prevacuolar transport and with the cis-Golgi t-SNARE Sed5p in traffic to the cis-Golgi. Here we describe a human Vti1p homolog, hVti1. Whereas vti1Delta cells are inviable, expression of hVti1 allows vti1Delta cells to grow at nearly the wild-type growth rate. When expressed in yeast hVti1 can replace Vti1p in both Golgi to prevacuolar transport and in traffic to the cis-Golgi. Sequence comparisons with a Schizosaccharomyces pombe and two different mouse Vti1 homologs led to the identification of a very conserved predicted alpha-helix. Amino acid exchanges in vti1 mutant alleles defective either in one or both trafficking steps cluster in this domain, suggesting that this structure is probably the binding site for effector proteins.


Subject(s)
Carrier Proteins/metabolism , Golgi Apparatus/metabolism , Intracellular Membranes/metabolism , Membrane Proteins/metabolism , Amino Acid Sequence , Animals , Biological Transport , Carrier Proteins/genetics , Conserved Sequence , Fungal Proteins/genetics , Genes, Lethal , Genetic Complementation Test , Humans , Membrane Proteins/genetics , Mice , Models, Molecular , Molecular Sequence Data , Protein Structure, Secondary , Qb-SNARE Proteins , Schizosaccharomyces/genetics , Sequence Homology, Amino Acid , Suppression, Genetic
4.
Eur J Cell Biol ; 65(2): 319-26, 1994 Dec.
Article in English | MEDLINE | ID: mdl-7720727

ABSTRACT

After exocytosis, synaptic vesicles rapidly endocytose and recycle but little is known about the molecular mechanisms involved. Rab5 is a ubiquitous low molecular weight GTP-binding protein required for endosomal fusion in fibroblasts. We have now raised polyclonal and monoclonal antibodies to rat Rab5 and show that in rat brain, Rab5 is a major synaptic vesicle protein. Immunoisolation of vesicular organelles from brain with antibodies to either Rab3A and Rab5 as small GTP-binding proteins or with synaptophysin as general synaptic vesicle marker demonstrates that there are overlapping populations of synaptic vesicles containing either Rab5 or Rab3A or both, suggesting a stage-specific association of these low-molecular weight GTP-binding proteins with synaptic vesicles. Our data provide the first biochemical evidence that synaptic vesicle recycling involves an endosomal intermediate similar to that of the receptor-mediated endocytosis pathway.


Subject(s)
Endosomes/metabolism , GTP-Binding Proteins/analysis , Nerve Tissue Proteins/analysis , Synaptic Vesicles/chemistry , Amino Acid Sequence , Base Sequence , Biomarkers/chemistry , Endocytosis/physiology , Molecular Sequence Data , Molecular Weight , rab3 GTP-Binding Proteins , rab5 GTP-Binding Proteins
5.
Trends Biochem Sci ; 19(4): 164-8, 1994 Apr.
Article in English | MEDLINE | ID: mdl-8016866

ABSTRACT

Regulated exocytosis is responsible for neuronal communication, hormone secretion, food digestion, control of glucose uptake and many other basic processes. Despite the structural and functional diversity of the cells undergoing regulated exocytosis, all regulated exocytosis involves specialized vesicles that are stored in the cytoplasm and fuse with the plasma membrane in response to a trigger event. Recent evidence suggests that a subset of small GTP-binding proteins, Rab3 and its relatives, participate in the control of regulated exocytosis.


Subject(s)
Exocytosis , GTP-Binding Proteins/physiology , Animals , Brain Chemistry , Cytoplasmic Granules/chemistry , Cytoplasmic Granules/physiology , GTP-Binding Proteins/analysis , Neurons/chemistry , Neurons/physiology , rab3 GTP-Binding Proteins
6.
J Biol Chem ; 269(15): 10971-4, 1994 Apr 15.
Article in English | MEDLINE | ID: mdl-8157621

ABSTRACT

Rab3 proteins are small GTP-binding proteins of the Ras superfamily. Four highly homologous Rab3 proteins termed Rab3A, Rab3B, Rab3C, and Rab3D have been described. Rab3A has previously been shown to be a constituent of synaptic vesicles in neurons that undergoes membrane dissociation-association cycles during synaptic vesicle recycling. Here we report that Rab3C copurifies with Rab3A during the isolation of synaptic vesicles. Organelles immunoisolated with monoclonal antibodies directed against Rab3A led to a coenrichment of Rab3A and Rab3C, demonstrating that both Rab3 proteins are colocalized on the same organelle. In isolated nerve terminals, stimulation of neurotransmitter release resulted in a dissociation of Rab3C from synaptic vesicle and/or recycling membranes. This dissociation parallels that of Rab3A observed under the same conditions. In contrast, no change was observed in the membrane-association of Rab5, a Rab protein localized on early endosomes. We conclude that in the nervous system Rab3C is localized on synaptic vesicles and, like Rab3A, cycles on and off the synaptic vesicle membrane in parallel with exocytotic release of neurotransmitter.


Subject(s)
Cerebral Cortex/metabolism , Exocytosis , GTP-Binding Proteins/metabolism , Nerve Tissue Proteins/metabolism , Synaptic Vesicles/metabolism , Amino Acid Sequence , Animals , Antibodies , Base Sequence , Cell Line , Cloning, Molecular , DNA Primers , GTP-Binding Proteins/biosynthesis , GTP-Binding Proteins/isolation & purification , Immunoblotting , Molecular Sequence Data , Organelles/metabolism , Peptides/chemical synthesis , Peptides/immunology , Polymerase Chain Reaction , Rabbits/immunology , Rats , Transfection , rab3 GTP-Binding Proteins
7.
J Biol Chem ; 268(25): 18423-6, 1993 Sep 05.
Article in English | MEDLINE | ID: mdl-8360142

ABSTRACT

Tetanus toxin is a potent inhibitor of neurotransmitter release, which acts as an intracellular metalloendoprotease that selectively cleaves synaptobrevin, a major membrane protein of synaptic vesicles. Recently, synaptobrevin has been found to form an ATP-dependent complex with N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment protein, which are known to function in endosome fusion. Furthermore, a highly homologous isoform of synaptobrevin, named cellubrevin, was identified that is expressed in virtually all tissues in the endocytic pathway and is cleaved by tetanus toxin light chain in vitro, suggesting that cellubrevin may have a general function in intracellular fusion events. In the present study, we have analyzed whether cleavage of cellubrevin by tetanus toxin influences the ATP-dependent, N-ethylmaleimide-sensitive fusion of early endosomes in vitro. Our results show that endosome fusion is not affected by tetanus toxin although cellubrevin is almost completely proteolyzed, suggesting that the function of NSF in endosome fusion does not involve cellubrevin.


Subject(s)
Endocytosis , Intracellular Membranes/physiology , Membrane Fusion , Membrane Proteins/metabolism , Tetanus Toxin/metabolism , Vesicular Transport Proteins , Adenosine Triphosphate/pharmacology , Animals , Carrier Proteins/metabolism , Cell Line , Cricetinae , Ethylmaleimide/pharmacology , Kidney , Kinetics , N-Ethylmaleimide-Sensitive Proteins , Nerve Tissue Proteins/metabolism , Organelles/ultrastructure , R-SNARE Proteins , Vesicle-Associated Membrane Protein 3
8.
Biochem J ; 293 ( Pt 1): 157-63, 1993 Jul 01.
Article in English | MEDLINE | ID: mdl-7687127

ABSTRACT

Rab3A is a neuronal low-molecular-mass GTP-binding protein that is modified post-translationally by two geranylgeranyl groups and specifically targeted to synaptic vesicles. We have now cloned and characterized the murine gene coding for rab3A. With a size of less than 8 kb including the promoter, the rab3A gene is relatively small. It contains five exons, the first of which is non-coding. The organization of the rab3A coding sequence into exons in the gene is different from that of ras proteins, the only other low-molecular-mass GTP-binding proteins with currently characterized gene structures. Nevertheless, the intron placement in the primary structure of rab3A may be indicative of a domain division of the protein, since each coding exon contains one of the four major conserved rab protein sequence motifs. The epitopes of monoclonal and polyclonal antibodies to rab3A were mapped with the hypothesis that antibody epitopes might represent distinct exposed protein domains and correlate with exon structures. Two monoclonal antibodies, named 42.1 and 42.2, were found to recognize epitopes with a different degree of conservation between different rab3 isoforms. These epitopes were mapped to relatively short amino acid sequences corresponding to exons 4 and 5 respectively, whereas a polyclonal antibody recognized a complex epitope that required the presence of intact rab3A. Comparison of the sequence of rab3A with that of ras, whose crystal structure has been determined, revealed that the epitopes for the monoclonal antibodies correspond to regions in ras that are highly exposed. Taken together, these results suggest that exons 4 and 5 at least represent distinct exposed protein domains that also form major natural epitopes in rab3A.


Subject(s)
Epitopes/immunology , GTP-Binding Proteins/genetics , Nerve Tissue Proteins/genetics , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Base Sequence , Cloning, Molecular , Epitopes/genetics , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/immunology , Gene Expression , Mice , Molecular Sequence Data , Molecular Weight , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/immunology , Plasmids , rab3 GTP-Binding Proteins
9.
Nature ; 349(6304): 79-81, 1991 Jan 03.
Article in English | MEDLINE | ID: mdl-1845915

ABSTRACT

Low-molecular-weight GTP-binding proteins are strong candidates for regulators of membrane traffic. In yeast, mutations in the sec4 or ypt1 genes encoding small GTP-binding proteins inhibit constitutive membrane flow at the plasma membrane or Golgi complex, respectively. It has been suggested that membrane fusion-fission events are regulated by cycling of small GTP-binding proteins between a membrane-bound and free state, but although most of these small proteins are found in both soluble and tightly membrane-bound forms, there is no direct evidence to support such cycling. In rat brain a small GTP-binding protein, rab3A, is exclusively associated with synaptic vesicles, the secretory organelles of nerve terminals. Here we use isolated nerve terminals to study the fate of rab3A during synaptic vesicle exocytosis. We find that rab3A dissociates quantitatively from the vesicle membrane after Ca2(+)-dependent exocytosis and that this dissociation is partially reversible during recovery after stimulation. These results are direct evidence for an association-dissociation cycle of a small GTP-binding protein during traffic of its host membrane.


Subject(s)
Exocytosis , GTP-Binding Proteins/metabolism , Nerve Tissue Proteins/metabolism , Synaptic Vesicles/metabolism , Animals , Molecular Weight , Rats , rab3 GTP-Binding Proteins
10.
J Cell Biol ; 110(4): 1285-94, 1990 Apr.
Article in English | MEDLINE | ID: mdl-2182650

ABSTRACT

A novel membrane protein from rat brain synaptic vesicles with an apparent 29,000 Mr (p29) was characterized. Using monospecific polyclonal antibodies, the distribution of p29 was studied in a variety of tissues by light and electron microscopy and immunoblot analysis. Within the nervous system, p29 was present in virtually all nerve terminals. It was selectively associated with small synaptic vesicles and a perinuclear region corresponding to the area of the Golgi complex. P29 was not detected in any other subcellular organelles including large dense-core vesicles. The distribution of p29 in various subcellular fractions from rat brain was very similar to that of synaptophysin and synaptobrevin. The highest enrichment occurred in purified small synaptic vesicles. Outside the nervous system, p29 was found only in endocrine cell types specialized for peptide hormone secretion. In these cells, p29 had a distribution very similar to that of synaptophysin. It was associated with microvesicles of heterogeneous size and shape that are primarily concentrated in the centrosomal-Golgi complex area. Secretory granules were mostly unlabeled, but their membrane occasionally contained small labeled evaginations. Immunoisolation of subcellular organelles from undifferentiated PC12 cells with antisynaptophysin antibodies led to a concomitant enrichment of p29, synaptobrevin, and synaptophysin, further supporting a colocalization of all three proteins. P29 has an isoelectric point of approximately 5.0 and is not N-glycosylated. It is an integral membrane protein and all antibody binding sites are exposed on the cytoplasmic side of the vesicles. Two monoclonal antibodies raised against p29 cross reacted with synaptophysin, indicating the presence of related epitopes. P29, like synaptophysin, was phosphorylated on tyrosine residues by endogenous tyrosine kinase activity in intact vesicles.


Subject(s)
Brain/cytology , Endocrine Glands/cytology , Membrane Proteins/analysis , Nerve Tissue Proteins/analysis , Neurons/cytology , Organelles/ultrastructure , Phosphoproteins/analysis , Animals , Antibodies , Antibodies, Monoclonal , Brain/ultrastructure , Brain Chemistry , Cattle , Cell Fractionation , Cerebral Cortex/analysis , Cerebral Cortex/cytology , Cerebral Cortex/ultrastructure , Electrophoresis, Polyacrylamide Gel , Endocrine Glands/analysis , Endocrine Glands/ultrastructure , Fluorescent Antibody Technique , Microscopy, Electron , Molecular Weight , Motor Endplate/cytology , Motor Endplate/ultrastructure , Neurons/analysis , Neurons/ultrastructure , Organ Specificity , Rats , Synapses/ultrastructure
11.
Proc Natl Acad Sci U S A ; 87(5): 1988-92, 1990 Mar.
Article in English | MEDLINE | ID: mdl-2155429

ABSTRACT

rab3, a low molecular weight GTP-binding protein, is primarily expressed in brain, where it is present in soluble and membrane-bound forms. Membrane-bound rab3 in brain is exclusively localized on synaptic vesicles, the secretory organelles of the synapse that store and release neurotransmitters. rab3 is also expressed in endocrine tissues such as the adrenal medulla, where it is found together with other synaptic vesicle proteins on microvesicles distinct from chromaffin granules. The tight binding of rab3 to membranes correlates with hydrophobic modifications that are different in the membrane-bound and soluble forms of rab3. The results demonstrate the exclusive targeting of a small GTP-binding protein to secretory vesicles of a subset of the regulated pathway of secretion.


Subject(s)
GTP-Binding Proteins/metabolism , Nerve Tissue Proteins/metabolism , Synaptic Vesicles/metabolism , Amino Acid Sequence , Animals , Antibodies , Cell Fractionation , Cerebral Cortex/metabolism , Chromaffin Granules/metabolism , Electrophoresis, Polyacrylamide Gel , GTP-Binding Proteins/isolation & purification , Molecular Sequence Data , Molecular Weight , Nerve Tissue Proteins/isolation & purification , Protein Binding , Rats , rab3 GTP-Binding Proteins
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