Export of Cellubrevin from the Endoplasmic Reticulum Is Controlled by BAP31

Cellubrevin is a ubiquitously expressed membrane protein that is localized to endosomes throughout the endocytotic pathway and functions in constitutive exocytosis. We report that cellubrevin binds with high specificity to BAP31, a representative of a highly conserved family of integral membrane proteins that has recently been discovered to be binding proteins of membrane immunoglobulins. The interaction between BAP31 and cellubrevin is sensitive to high ionic strength and appears to require the transmembrane regions of both proteins. No other proteins of liver membrane extracts copurified with BAP31 on immobilized recombinant cellubrevin, demonstrating that the interaction is specific. Synaptobrevin I bound to BAP31 with comparable affinity, whereas only weak binding was detectable with synaptobrevin II. Furthermore, a fraction of BAP31 and cellubrevin was complexed when each of them was quantitatively immunoprecipitated from detergent extracts of fibroblasts (BHK 21 cells). During purification of clathrin-coated vesicles or early endosomes, BAP31 did not cofractionate with cellubrevin. Rather, the protein was enriched in ER-containing fractions. When BHK cells were analyzed by immunocytochemistry, BAP31 did not overlap with cellubrevin, but rather colocalized with resident proteins of the ER. In addition, immunoreactive vesicles were clustered in a paranuclear region close to the microtubule organizing center, but different from the Golgi apparatus. When microtubules were depolymerized with nocodazole, this accumulation disappeared and BAP31 was confined to the ER. Truncation of the cytoplasmic tail of BAP31 prevented export of cellubrevin, but not of the transferrin receptor from the ER. We conclude that BAP31 represents a novel class of sorting proteins that controls anterograde transport of certain membrane proteins from the ER to the Golgi complex.Exocytotic membrane fusion is mediated by a complex of evolutionary-conserved membrane proteins. In neurons, these proteins include the synaptic vesicle protein synaptobrevin (VAMP) and the synaptic membrane proteins syntaxin and synaptosome-associated protein (SNAP)-25.¹ These proteins undergo regulated protein–protein interactions that are controlled by soluble proteins including N-ethylmaleimide-sensitive factor (NSF) and soluble N-ethyl maleimide-sensitive factor attachment (SNAP) proteins (Söllner et al., 1993b ). Relatives of all of these proteins have been discovered in many eukaryotic cells including yeast, suggesting that intracellular membrane fusions may, at least to a large extent, be mediated by common mechanisms (Ferro-Novick and Jahn, 1994; Rothman, 1994; Scheller, 1995). Although the molecular details of membrane fusion are not yet understood, it is becoming clear that the components of the fusion apparatus operate by conformation-dependent assembly and disassembly reactions which ultimately lead to the rearrangement of membrane phospholipids (Söllner et al., 1993a ; Calakos et al., 1994). For these reasons, the interactions between synaptobrevin, SNAP-25, and syntaxin have received considerable attention (for review see Südhof, 1995). These proteins form a tight and stable ternary complex as soon as they have access to each other. Binding probably occurs before or during vesicle docking in preparation for fusion. Incubation with the ATPase NSF and SNAP proteins reversibly disassembles this complex, an event thought to precede membrane fusion (Söllner et al., 1993a ,b).It is less well understood to what extent synaptobrevin, SNAP-25, and syntaxin interact with other proteins, particularly during stages of their life cycle when they are not bound to each other. It is conceivable that companion proteins exist that assist in sorting to the correct compartment or in positioning at the site of release and that control the availability for entering the fusion complex. For syntaxin, interactions with several other proteins were reported, including synaptotagmin munc-18/rbSEC-1, and the N-type Ca²⁺-channel (Südhof, 1995). For synaptobrevin, it has recently been observed that most of the protein is associated with synaptophysin, an integral membrane protein of yet unknown function that resides alongside synaptobrevin in the synaptic vesicle membrane (Calakos and Scheller, 1994; Edelmann et al., 1995; Washbourne et al., 1995). Although the binary interaction of synaptobrevin with synaptophysin is weaker than its ternary interaction with syntaxin and SNAP-25, synaptophysin-bound synaptobrevin is not available for binding to these proteins (Edelmann et al., 1995). Thus, synaptobrevin participates at least in two different complexes that are mutually exclusive: one with its partners syntaxin and SNAP-25 during membrane fusion, and another with synaptophysin during vesicle recycling and probably also during biogenesis, i.e., during transport of the proteins from the ER to the nerve terminal.It remains to be established whether cellubrevin, a nonneuronal synaptobrevin homologue with widespread distribution, forms partnerships with other proteins with properties similar to the synaptobrevin–synaptophysin complex. Like synaptobrevins, cellubrevin is a small integral membrane protein with a single transmembrane domain at the COOH-terminal end of the molecule. Cellubrevin colocalizes with the transferrin receptor in fibroblasts and is enriched in purified clathrin-coated vesicles (McMahon et al., 1993), suggesting that it resides in constitutive trafficking vesicles shuttling mainly between the plasmalemma and the endosomal compartment (Daro et al., 1996). Like its neuronal counterparts, cellubrevin is selectively cleaved by clostridial neurotoxins including tetanus toxin. Toxin cleavage impairs exocytosis of recycling vesicles in fibroblasts (Galli et al., 1994), whereas fusion of early endosomes appears not to be affected (Link et al., 1993; Jo et al., 1995).Here we report that cellubrevin interacts specifically with a recently characterized integral membrane protein, BAP31. BAP31 and a related protein (BAP29) were first identified as membrane proteins copurifying with membrane-bound immunoglobulin from lysates of β lymphocytes (Kim et al., 1994). Cloning of human and murine BAP31 cDNA showed that BAP31 is an evolutionary-conserved protein which is ubiquitously expressed in all tissues (Adachi et al., 1996). Several open reading frames encoding for proteins with a similar structure and a significant degree of homology are present in the genome of the yeast Saccharomyces cerevisiae, suggesting that BAP31 represents an ancient protein family with basic functions (EMBL/GenBank/DDBJ accession numbers {"type":"entrez-nucleotide","attrs":{"text":"Z28065","term_id":"486088","term_text":"Z28065"}}Z28065, {"type":"entrez-nucleotide","attrs":{"text":"Z74120","term_id":"1431083","term_text":"Z74120"}}Z74120, and {"type":"entrez-nucleotide","attrs":{"text":"Z48502","term_id":"695715","term_text":"Z48502"}}Z48502). BAP31 has a hydrophobic NH₂ terminus with three potential transmembrane domains and a charged α-helical COOH terminus that is exposed to the cytoplasm. The COOH terminus ends with a KKXX sequence motif typical for proteins transported back to the ER. Indeed, an immunocytochemical analysis revealed that BAP31 exhibits an ER-like staining pattern (Becker, B., and M. Reth, unpublished observations). We show that BAP31, as a resident of the ER and of ER-derived trafficking vesicles, may control the export of cellubrevin from the ER.