Identification of a cellubrevin/vesicle associated membrane protein 3 homologue in human platelets.
Several studies suggest membrane trafficking events are mediated by integral, membrane proteins from both transport-vesicle and target membranes, called v- and t-SNAREs (SNAp REceptors), respectively. Previous experiments using antibodies to synaptobrevin/vesicle associated membrane protein (VAMP) 1, 2, or rat cellubrevin failed to detect these v-SNAREs in human platelets, although membrane proteins from these cells could support 20S complex formation. To identify v-SNAREs in platelets, we used a polymerase chain reaction (PCR) approach with degenerate primers to amplify potential VAMP-like v-SNAREs. A cDNA encoding a novel v-SNARE was isolated from a human megakaryocyte cDNA library. Termed human cellubrevin (Hceb), this protein has greater than 93% identity with human VAMP 1, 2, and rat cellubrevin over the conserved core region, but has a unique N-terminal domain. Northern blot analysis showed that the 2. 5-kB mRNA encoding Hceb is expressed in every human tissue tested. Hceb from detergent-solubilized platelet membranes, participated in alpha-SNAP-dependent 20S complex formation and adenosine triphosphate (ATP)-dependent disassembly, showing that Hceb can act as a v-SNARE in platelets. Immunofluorescence microscopy, using an anti-Hceb antibody showed a punctate, intracellular staining pattern in platelets, megakaryocytes, and HEK-293 cells. This same pattern was observed in surface-activated platelets even though all dense core and most alpha-granule contents had been released. These data suggest that Hceb may reside on a platelet organelle that is not primarily involved in the exocytic pathway.
Bernstein
A M
AM
Department of Biochemistry, University of Kentucky College of Medicine, Chandler Medical Center, Lexington, KY 40536-0084, USA.
Whiteheart
S W
SW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001792:Blood Platelets / Q000737:chemistry*; D015152:Blotting, Northern; D018076:DNA, Complementary / Q000032:analysis / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D006801:Humans; D008533:Megakaryocytes / Q000737:chemistry; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017385:Sequence Homology; D014018:Tissue Distribution; D051040:Vesicle-Associated Membrane Protein 3
D018076:DNA, Complementary; D008565:Membrane Proteins; D012333:RNA, Messenger; D051040:Vesicle-Associated Membrane Protein 3