Dataset Information

Endobrevin, a novel synaptobrevin/VAMP-like protein preferentially associated with the early endosome.

ABSTRACT: Synaptobrevins/vesicle-associated membrane proteins (VAMPs) together with syntaxins and a synaptosome-associated protein of 25 kDa (SNAP-25) are the main components of a protein complex involved in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, biochemical, and cell biological characterization of a novel member of the synaptobrevin/VAMP family. The amino acid sequence of endobrevin has 32, 33, and 31% identity to those of synaptobrevin/VAMP-1, synaptobrevin/VAMP-2, and cellubrevin, respectively. Membrane fractionation studies demonstrate that endobrevin is enriched in membrane fractions that are also enriched in the asialoglycoprotein receptor. Indirect immunofluorescence microscopy establishes that endobrevin is primarily associated with the perinuclear vesicular structures of the early endocytic compartment. The preferential association of endobrevin with the early endosome was further established by electron microscopy (EM) immunogold labeling. In vitro binding assays show that endobrevin interacts with immobilized recombinant alpha-SNAP fused to glutathione S-transferase (GST). Our results highlight the general importance of members of the synaptobrevin/VAMP protein family in membrane traffic and provide new avenues for future functional and mechanistic studies of this protein as well as the endocytotic pathway.

SUBMITTER: Wong SH

PROVIDER: S-EPMC25382 | biostudies-literature | 1998 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Endobrevin, a novel synaptobrevin/VAMP-like protein preferentially associated with the early endosome.

Wong S H SH Zhang T T Xu Y Y Subramaniam V N VN Griffiths G G Hong W W

Molecular biology of the cell 19980601 6

Synaptobrevins/vesicle-associated membrane proteins (VAMPs) together with syntaxins and a synaptosome-associated protein of 25 kDa (SNAP-25) are the main components of a protein complex involved in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, biochemical, and cell biological characterization of a novel member of the synaptobrevin/VAMP family. The amino acid sequence of endobrevin has 32, 33, and 31% identity to those of synaptobrevin ...[more]

PMID: 9614193

Similar Datasets

Project description:Molecular studies have identified a family of synaptic vesicle-associated membrane proteins (VAMPs, also known as synaptobrevins) which have been implicated in synaptic vesicle docking and/or fusion with plasma membrane proteins. Here we demonstrate the expression of two members of this family, VAMP-2/synaptobrevin II and cellubrevin, in skeletal muscle, a tissue with both constitutive and regulated membrane traffic. The 18 kDa VAMP-2 polypeptide was detected in purified membrane fractions from adult skeletal muscle and from L6 myotubes in culture, demonstrating that the presence of this protein in the isolated muscle membrane fractions is not the result of contamination by ancillary tissues such as peripheral nerve. Furthermore, skeletal muscle and the muscle cell line also expressed cellubrevin, a VAMP-2 homologue of 17 kDa; which is much less abundant in brain cells. Both VAMP-2 and cellubrevin were preferentially isolated in membrane fractions rich in plasma membranes, and were less concentrated in light microsomes and other internal membrane fractions of mature muscle or muscle cells in culture. Interestingly, both VAMP-2 and cellubrevin were much more abundant in the differentiated L6 myotubes than in their precursor myoblasts, suggesting that they are required for functions of differentiated muscle cells. The identity of both polypeptides was further confirmed by their susceptibility to proteolysis by Clostridium tetanus toxin. Expression of these products was further established by the presence of mRNA transcripts of VAMP-2 and cellubrevin, but not of VAMP-1, in both skeletal muscle and L6 myotubes. In contrast, other synaptic vesicle and docking/fusion components were undetectable, such as VAMP-1, SNAP25 and syntaxin 1A/1B, as were synaptophysin and synapsin Ia/Ib, proteins which are believed to be involved in sensing the signal for neuronal exocytosis. It is concluded that VAMP-2 and cellubrevin are expressed in skeletal muscle cells and may each participate in specific processes of intracellular membrane traffic.

Project description:BACKGROUND: The control of intracellular vesicle trafficking is an ideal target to weigh the role of alternative splicing in shaping genomes to make cells. Alternative splicing has been reported for several Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptors of the vesicle (v-SNAREs) or of the target membrane (t-SNARES), which are crucial to intracellular membrane fusion and protein and lipid traffic in Eukaryotes. However, splicing has not yet been investigated in Longins, i.e. the most widespread v-SNAREs. Longins are essential in Eukaryotes and prototyped by VAMP7, Sec22b and Ykt6, sharing a conserved N-terminal Longin domain which regulates membrane fusion and subcellular targeting. Human VAMP7/TI-VAMP, encoded by gene SYBL1, is involved in multiple cell pathways, including control of neurite outgrowth. RESULTS: Alternative splicing of SYBL1 by exon skipping events results in the production of a number of VAMP7 isoforms. In-frame or frameshift coding sequence modifications modulate domain architecture of VAMP7 isoforms, which can lack whole domains or domain fragments and show variant or extra domains. Intriguingly, two main types of VAMP7 isoforms either share the inhibitory Longin domain and lack the fusion-promoting SNARE motif, or vice versa. Expression analysis in different tissues and cell lines, quantitative real time RT-PCR and confocal microscopy analysis of fluorescent protein-tagged isoforms demonstrate that VAMP7 variants have different tissue specificities and subcellular localizations. Moreover, design and use of isoform-specific antibodies provided preliminary evidence for the existence of splice variants at the protein level. CONCLUSIONS: Previous evidence on VAMP7 suggests inhibitory functions for the Longin domain and fusion/growth promoting activity for the ?-longin molecule. Thus, non-SNARE isoforms with Longin domain and non-longin SNARE isoforms might have somehow opposite regulatory functions. When considering splice variants as "natural mutants", evidence on modulation of subcellular localization by variation in domain combination can shed further light on targeting determinants. Although further work will be needed to characterize identified variants, our data might open the route to unravel novel molecular partners and mechanisms, accounting for the multiplicity of functions carried out by the different members of the Longin proteins family.

Dataset Information

Endobrevin, a novel synaptobrevin/VAMP-like protein preferentially associated with the early endosome.

Publications

Endobrevin, a novel synaptobrevin/VAMP-like protein preferentially associated with the early endosome.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets