Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1995 Jan 16;14(2):217–223. doi: 10.1002/j.1460-2075.1995.tb06994.x

Transport route for synaptobrevin via a novel pathway of insertion into the endoplasmic reticulum membrane.

U Kutay 1, G Ahnert-Hilger 1, E Hartmann 1, B Wiedenmann 1, T A Rapoport 1
PMCID: PMC398073  PMID: 7835332

Abstract

Synaptobrevin/vesicle-associated membrane protein is one of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. It is proposed to provide specificity for the targeting and fusion of vesicles with the plasma membrane. It belongs to a class of membrane proteins which lack a signal sequence and contain a single hydrophobic segment close to their C-terminus, leaving most of the polypeptide chain in the cytoplasm (tail-anchored). We show that in neuroendocrine PC12 cells, synaptobrevin is not directly incorporated into the target organelle, synaptic-like vesicles. Rather, it is first inserted into the endoplasmic reticulum (ER) membrane and is then transported via the Golgi apparatus. Its insertion into the ER membrane in vitro occurs post-translationally, is dependent on ATP and results in a trans-membrane orientation of the hydrophobic tail. Membrane integration requires ER protein(s) different from the translocation components needed for proteins with signal sequences, thus suggesting a novel mechanism of insertion.

Full text

PDF
217

Images in this article

218Image
on p.218
219Image
on p.219
220Image
on p.220
220Image
on p.220
221Image
on p.221
221Image
on p.221

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andrews D. W., Gupta J., Abisdris G. Evidence that the middle T antigen of polyomavirus interacts with the membrane skeleton. Mol Cell Biol. 1993 Aug;13(8):4703–4713. doi: 10.1128/mcb.13.8.4703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andrews D. W., Lauffer L., Walter P., Lingappa V. R. Evidence for a two-step mechanism involved in assembly of functional signal recognition particle receptor. J Cell Biol. 1989 Mar;108(3):797–810. doi: 10.1083/jcb.108.3.797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Archer B. T., 3rd, Ozçelik T., Jahn R., Francke U., Südhof T. C. Structures and chromosomal localizations of two human genes encoding synaptobrevins 1 and 2. J Biol Chem. 1990 Oct 5;265(28):17267–17273. [PubMed] [Google Scholar]
  4. Arinç E., Rzepecki L. M., Strittmatter P. Topography of the C terminus of cytochrome b5 tightly bound to dimyristoylphosphatidylcholine vesicles. J Biol Chem. 1987 Nov 15;262(32):15563–15567. [PubMed] [Google Scholar]
  5. Banfield D. K., Lewis M. J., Rabouille C., Warren G., Pelham H. R. Localization of Sed5, a putative vesicle targeting molecule, to the cis-Golgi network involves both its transmembrane and cytoplasmic domains. J Cell Biol. 1994 Oct;127(2):357–371. doi: 10.1083/jcb.127.2.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Baumert M., Maycox P. R., Navone F., De Camilli P., Jahn R. Synaptobrevin: an integral membrane protein of 18,000 daltons present in small synaptic vesicles of rat brain. EMBO J. 1989 Feb;8(2):379–384. doi: 10.1002/j.1460-2075.1989.tb03388.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bennett M. K., Scheller R. H. The molecular machinery for secretion is conserved from yeast to neurons. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2559–2563. doi: 10.1073/pnas.90.7.2559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Borgese N., D'Arrigo A., De Silvestris M., Pietrini G. NADH-cytochrome b5 reductase and cytochrome b5. The problem of posttranslational targeting to the endoplasmic reticulum. Subcell Biochem. 1993;21:313–341. doi: 10.1007/978-1-4615-2912-5_14. [DOI] [PubMed] [Google Scholar]
  9. Calakos N., Bennett M. K., Peterson K. E., Scheller R. H. Protein-protein interactions contributing to the specificity of intracellular vesicular trafficking. Science. 1994 Feb 25;263(5150):1146–1149. doi: 10.1126/science.8108733. [DOI] [PubMed] [Google Scholar]
  10. Dailey H. A., Strittmatter P. Structural and functional properties of the membrane binding segment of cytochrome b5. J Biol Chem. 1978 Nov 25;253(22):8203–8209. [PubMed] [Google Scholar]
  11. Feldheim D., Schekman R. Sec72p contributes to the selective recognition of signal peptides by the secretory polypeptide translocation complex. J Cell Biol. 1994 Aug;126(4):935–943. doi: 10.1083/jcb.126.4.935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Green N., Fang H., Walter P. Mutants in three novel complementation groups inhibit membrane protein insertion into and soluble protein translocation across the endoplasmic reticulum membrane of Saccharomyces cerevisiae. J Cell Biol. 1992 Feb;116(3):597–604. doi: 10.1083/jcb.116.3.597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Görlich D., Prehn S., Hartmann E., Kalies K. U., Rapoport T. A. A mammalian homolog of SEC61p and SECYp is associated with ribosomes and nascent polypeptides during translocation. Cell. 1992 Oct 30;71(3):489–503. doi: 10.1016/0092-8674(92)90517-g. [DOI] [PubMed] [Google Scholar]
  14. Görlich D., Rapoport T. A. Protein translocation into proteoliposomes reconstituted from purified components of the endoplasmic reticulum membrane. Cell. 1993 Nov 19;75(4):615–630. doi: 10.1016/0092-8674(93)90483-7. [DOI] [PubMed] [Google Scholar]
  15. Hartmann E., Görlich D., Kostka S., Otto A., Kraft R., Knespel S., Bürger E., Rapoport T. A., Prehn S. A tetrameric complex of membrane proteins in the endoplasmic reticulum. Eur J Biochem. 1993 Jun 1;214(2):375–381. doi: 10.1111/j.1432-1033.1993.tb17933.x. [DOI] [PubMed] [Google Scholar]
  16. Hortsch M., Meyer D. I. The human docking protein does not associate with the membrane of the rough endoplasmic reticulum via a signal or insertion sequence-mediated mechanism. Biochem Biophys Res Commun. 1988 Jan 15;150(1):111–117. doi: 10.1016/0006-291x(88)90493-7. [DOI] [PubMed] [Google Scholar]
  17. Janiak F., Leber B., Andrews D. W. Assembly of Bcl-2 into microsomal and outer mitochondrial membranes. J Biol Chem. 1994 Apr 1;269(13):9842–9849. [PubMed] [Google Scholar]
  18. Klappa P., Zimmermann M., Zimmermann R. The membrane proteins TRAMp and sec61 alpha p may be involved in post-translational transport of presecretory proteins into mammalian microsomes. FEBS Lett. 1994 Mar 21;341(2-3):281–287. doi: 10.1016/0014-5793(94)80473-7. [DOI] [PubMed] [Google Scholar]
  19. Klausner R. D., Donaldson J. G., Lippincott-Schwartz J. Brefeldin A: insights into the control of membrane traffic and organelle structure. J Cell Biol. 1992 Mar;116(5):1071–1080. doi: 10.1083/jcb.116.5.1071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kurihara T., Silver P. Suppression of a sec63 mutation identifies a novel component of the yeast endoplasmic reticulum translocation apparatus. Mol Biol Cell. 1993 Sep;4(9):919–930. doi: 10.1091/mbc.4.9.919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kutay U., Hartmann E., Rapoport T. A. A class of membrane proteins with a C-terminal anchor. Trends Cell Biol. 1993 Mar;3(3):72–75. doi: 10.1016/0962-8924(93)90066-a. [DOI] [PubMed] [Google Scholar]
  22. Migliaccio G., Nicchitta C. V., Blobel G. The signal sequence receptor, unlike the signal recognition particle receptor, is not essential for protein translocation. J Cell Biol. 1992 Apr;117(1):15–25. doi: 10.1083/jcb.117.1.15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mitoma J., Ito A. The carboxy-terminal 10 amino acid residues of cytochrome b5 are necessary for its targeting to the endoplasmic reticulum. EMBO J. 1992 Nov;11(11):4197–4203. doi: 10.1002/j.1460-2075.1992.tb05513.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nguyen M., Millar D. G., Yong V. W., Korsmeyer S. J., Shore G. C. Targeting of Bcl-2 to the mitochondrial outer membrane by a COOH-terminal signal anchor sequence. J Biol Chem. 1993 Dec 5;268(34):25265–25268. [PubMed] [Google Scholar]
  25. Nilsson I. M., von Heijne G. Determination of the distance between the oligosaccharyltransferase active site and the endoplasmic reticulum membrane. J Biol Chem. 1993 Mar 15;268(8):5798–5801. [PubMed] [Google Scholar]
  26. Noël P., Cartwright I. L. A Sec62p-related component of the secretory protein translocon from Drosophila displays developmentally complex behavior. EMBO J. 1994 Nov 15;13(22):5253–5261. doi: 10.1002/j.1460-2075.1994.tb06859.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ohno-Iwashita Y., Wickner W. Reconstitution of rapid and asymmetric assembly of M13 procoat protein into liposomes which have bacterial leader peptidase. J Biol Chem. 1983 Feb 10;258(3):1895–1900. [PubMed] [Google Scholar]
  28. Rapoport T. A. Transport of proteins across the endoplasmic reticulum membrane. Science. 1992 Nov 6;258(5084):931–936. doi: 10.1126/science.1332192. [DOI] [PubMed] [Google Scholar]
  29. Rothblatt J. A., Deshaies R. J., Sanders S. L., Daum G., Schekman R. Multiple genes are required for proper insertion of secretory proteins into the endoplasmic reticulum in yeast. J Cell Biol. 1989 Dec;109(6 Pt 1):2641–2652. doi: 10.1083/jcb.109.6.2641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schlenstedt G., Zimmermann R. Import of frog prepropeptide GLa into microsomes requires ATP but does not involve docking protein or ribosomes. EMBO J. 1987 Mar;6(3):699–703. doi: 10.1002/j.1460-2075.1987.tb04810.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sommer T., Jentsch S. A protein translocation defect linked to ubiquitin conjugation at the endoplasmic reticulum. Nature. 1993 Sep 9;365(6442):176–179. doi: 10.1038/365176a0. [DOI] [PubMed] [Google Scholar]
  32. Söllner T., Bennett M. K., Whiteheart S. W., Scheller R. H., Rothman J. E. A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. Cell. 1993 Nov 5;75(3):409–418. doi: 10.1016/0092-8674(93)90376-2. [DOI] [PubMed] [Google Scholar]
  33. Söllner T., Whiteheart S. W., Brunner M., Erdjument-Bromage H., Geromanos S., Tempst P., Rothman J. E. SNAP receptors implicated in vesicle targeting and fusion. Nature. 1993 Mar 25;362(6418):318–324. doi: 10.1038/362318a0. [DOI] [PubMed] [Google Scholar]
  34. Südhof T. C., Baumert M., Perin M. S., Jahn R. A synaptic vesicle membrane protein is conserved from mammals to Drosophila. Neuron. 1989 May;2(5):1475–1481. doi: 10.1016/0896-6273(89)90193-1. [DOI] [PubMed] [Google Scholar]
  35. Südhof T. C., De Camilli P., Niemann H., Jahn R. Membrane fusion machinery: insights from synaptic proteins. Cell. 1993 Oct 8;75(1):1–4. [PubMed] [Google Scholar]
  36. Takagaki Y., Radhakrishnan R., Wirtz K. W., Khorana H. G. The membrane-embedded segment of cytochrome b5 as studied by cross-linking with photoactivatable phospholipids. II. The nontransferable form. J Biol Chem. 1983 Aug 10;258(15):9136–9142. [PubMed] [Google Scholar]
  37. Walter P., Ibrahimi I., Blobel G. Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in-vitro-assembled polysomes synthesizing secretory protein. J Cell Biol. 1981 Nov;91(2 Pt 1):545–550. doi: 10.1083/jcb.91.2.545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wickner W., Driessen A. J., Hartl F. U. The enzymology of protein translocation across the Escherichia coli plasma membrane. Annu Rev Biochem. 1991;60:101–124. doi: 10.1146/annurev.bi.60.070191.000533. [DOI] [PubMed] [Google Scholar]
  39. Wiedmann M., Kurzchalia T. V., Bielka H., Rapoport T. A. Direct probing of the interaction between the signal sequence of nascent preprolactin and the signal recognition particle by specific cross-linking. J Cell Biol. 1987 Feb;104(2):201–208. doi: 10.1083/jcb.104.2.201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Yamasaki S., Hu Y., Binz T., Kalkuhl A., Kurazono H., Tamura T., Jahn R., Kandel E., Niemann H. Synaptobrevin/vesicle-associated membrane protein (VAMP) of Aplysia californica: structure and proteolysis by tetanus toxin and botulinal neurotoxins type D and F. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4688–4692. doi: 10.1073/pnas.91.11.4688. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES