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© The Rockefeller University Press, 0021-9525/1997//1397 $5.00
The Journal of Cell Biology, Volume 139, Number 6, , 1997 1397-1410

Article

Export of Cellubrevin from the Endoplasmic Reticulum Is Controlled by BAP31



Wim G. Annaert*, Bernd Becker{ddagger}, Ute Kistner*, Michael Reth{ddagger}, and Reinhard Jahn*

* Howard Hughes Medical Institute and Department of Pharmacology and Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510; and {ddagger} Max-Planck-Institut für Immunologie, D-79108 Freiburg, Germany

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.

Abbreviations used in this paper: aa, amino acids; AP, alkaline phosphatase; COP, coat proteins; DTAF, 5-(4,6-dichlorotrianzinyl) aminofluorescein; ECL, enhanced chemiluminescence; ERGIC, endoplasmic reticulum–Golgi intermediate compartment; MTOC, microtubule organizing center; PDI, protein disulfide isomerase; PNS, postnuclear supernatant; SCAMP, secretory carrier membrane proteins; SNAP, soluble NSF attachment protein; SNAP-25, synaptosome-associated protein of 25,000 kD.

W.G. Annaert's current address is Experimental Genetics Group, Center for Human Genetics, Herestraat 49, B-3000 Leuven, Belgium.

U. Kistner's current address is Institut für Anatomie, Medizinische Fakultät (Charite), Humboldt-Universität, Schumannstr. 20–22, D-10098 Berlin, Germany.

Address all correspondence to Reinhard Jahn, Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, D-37077 Göttingen, Germany. Tel.: (49) 551.201.1635. Fax: (49) 551.201.1639. E-mail: rjahn{at}gwdg.de

W.G. Annaert was supported by a D. Collen Fellowship and a Philips Fellowship of the Belgian American Educational Foundation, a Fulbright-Hays Award, and a North Atlantic Treaty Organization grant. U. Kistner was a recipient of a fellowship from the Boehringer-Ingelheim Foundation. This work was supported by the Deutsche Forschungsgemeinschaft by a grant to M. Reth through SFB364.


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