Abstract
We have characterized the process by which the vesicular stomatitis virus (VSV) G protein acquires its final oligomeric structure using density-gradient centrifugation in mildly acidic sucrose gradients. The mature wild-type VSV G protein is a noncovalently associated trimer. Trimers are assembled from newly synthesized G monomers with a t1/2 of 6-8 min. To localize the site of trimerization and to correlate trimer formation with steps in transport between the endoplasmic reticulum (ER) and Golgi complex, we examined the kinetics of assembly of the temperature-sensitive mutant VSV strain, ts045. At the nonpermissive temperature (39 degrees C), ts045 G protein is not transported from the ER. The phenotypic defect that inhibited export from the ER at the nonpermissive temperature was found to be the accumulation of ts045 G protein in an aggregate. After being shifted to the permissive temperature (32 degrees C), the ts045 G protein aggregate rapidly dissociated (t1/2 less than 1 min) to monomeric G protein which subsequently trimerized with the same kinetics as the wild-type G protein. Only trimers were transported to the Golgi complex. Kinetic studies, as well as the finding that trimerization occurred under conditions which block ER to Golgi transport (at both 15 and 4 degrees C), showed that trimers were formed in the ER. Depletion of cellular ATP inhibited both the dissociation of the aggregated intermediate of ts045 G protein as well as the formation of stable trimers. The results indicate that oligomerization of G protein occurs in several steps, is sensitive to cellular ATP, and is required for transport from the ER.
Full Text
The Full Text of this article is available as a PDF (2.5 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Balch W. E., Elliott M. M., Keller D. S. ATP-coupled transport of vesicular stomatitis virus G protein between the endoplasmic reticulum and the Golgi. J Biol Chem. 1986 Nov 5;261(31):14681–14689. [PubMed] [Google Scholar]
- Balch W. E., Keller D. S. ATP-coupled transport of vesicular stomatitis virus G protein. Functional boundaries of secretory compartments. J Biol Chem. 1986 Nov 5;261(31):14690–14696. [PubMed] [Google Scholar]
- Balch W. E., Wagner K. R., Keller D. S. Reconstitution of transport of vesicular stomatitis virus G protein from the endoplasmic reticulum to the Golgi complex using a cell-free system. J Cell Biol. 1987 Mar;104(3):749–760. doi: 10.1083/jcb.104.3.749. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Beckers C. J., Keller D. S., Balch W. E. Semi-intact cells permeable to macromolecules: use in reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex. Cell. 1987 Aug 14;50(4):523–534. doi: 10.1016/0092-8674(87)90025-0. [DOI] [PubMed] [Google Scholar]
- Bergmann J. E., Singer S. J. Immunoelectron microscopic studies of the intracellular transport of the membrane glycoprotein (G) of vesicular stomatitis virus in infected Chinese hamster ovary cells. J Cell Biol. 1983 Dec;97(6):1777–1787. doi: 10.1083/jcb.97.6.1777. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bole D. G., Hendershot L. M., Kearney J. F. Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas. J Cell Biol. 1986 May;102(5):1558–1566. doi: 10.1083/jcb.102.5.1558. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Copeland C. S., Doms R. W., Bolzau E. M., Webster R. G., Helenius A. Assembly of influenza hemagglutinin trimers and its role in intracellular transport. J Cell Biol. 1986 Oct;103(4):1179–1191. doi: 10.1083/jcb.103.4.1179. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Crimmins D. L., Mehard W. B., Schlesinger S. Physical properties of a soluble form of the glycoprotein of vesicular stomatitis virus at neutral and acidic pH. Biochemistry. 1983 Dec 6;22(25):5790–5796. doi: 10.1021/bi00294a017. [DOI] [PubMed] [Google Scholar]
- Doms R. W., Helenius A. Quaternary structure of influenza virus hemagglutinin after acid treatment. J Virol. 1986 Dec;60(3):833–839. doi: 10.1128/jvi.60.3.833-839.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Doms R. W., Helenius A., White J. Membrane fusion activity of the influenza virus hemagglutinin. The low pH-induced conformational change. J Biol Chem. 1985 Mar 10;260(5):2973–2981. [PubMed] [Google Scholar]
- Doyle C., Roth M. G., Sambrook J., Gething M. J. Mutations in the cytoplasmic domain of the influenza virus hemagglutinin affect different stages of intracellular transport. J Cell Biol. 1985 Mar;100(3):704–714. doi: 10.1083/jcb.100.3.704. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dubovi E. J., Wagner R. R. Spatial relationships of the proteins of vesicular stomatitis virus: induction of reversible oligomers by cleavable protein cross-linkers and oxidation. J Virol. 1977 May;22(2):500–509. doi: 10.1128/jvi.22.2.500-509.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dunphy W. G., Brands R., Rothman J. E. Attachment of terminal N-acetylglucosamine to asparagine-linked oligosaccharides occurs in central cisternae of the Golgi stack. Cell. 1985 Feb;40(2):463–472. doi: 10.1016/0092-8674(85)90161-8. [DOI] [PubMed] [Google Scholar]
- Gallione C. J., Rose J. K. A single amino acid substitution in a hydrophobic domain causes temperature-sensitive cell-surface transport of a mutant viral glycoprotein. J Virol. 1985 May;54(2):374–382. doi: 10.1128/jvi.54.2.374-382.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gething M. J., McCammon K., Sambrook J. Expression of wild-type and mutant forms of influenza hemagglutinin: the role of folding in intracellular transport. Cell. 1986 Sep 12;46(6):939–950. doi: 10.1016/0092-8674(86)90076-0. [DOI] [PubMed] [Google Scholar]
- Gibson R., Schlesinger S., Kornfeld S. The nonglycosylated glycoprotein of vesicular stomatitis virus is temperature-sensitive and undergoes intracellular aggregation at elevated temperatures. J Biol Chem. 1979 May 10;254(9):3600–3607. [PubMed] [Google Scholar]
- Gottlieb C., Baenziger J., Kornfeld S. Deficient uridine diphosphate-N-acetylglucosamine:glycoprotein N-acetylglucosaminyltransferase activity in a clone of Chinese hamster ovary cells with altered surface glycoproteins. J Biol Chem. 1975 May 10;250(9):3303–3309. [PubMed] [Google Scholar]
- Helenius A., Fries E., Garoff H., Simons K. Solubilization of the Semliki Forest virus membrane with sodium deoxycholate. Biochim Biophys Acta. 1976 Jun 17;436(2):319–334. doi: 10.1016/0005-2736(76)90197-8. [DOI] [PubMed] [Google Scholar]
- Hortsch M., Avossa D., Meyer D. I. Characterization of secretory protein translocation: ribosome-membrane interaction in endoplasmic reticulum. J Cell Biol. 1986 Jul;103(1):241–253. doi: 10.1083/jcb.103.1.241. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jamieson J. D., Palade G. E. Intracellular transport of secretory proteins in the pancreatic exocrine cell. IV. Metabolic requirements. J Cell Biol. 1968 Dec;39(3):589–603. doi: 10.1083/jcb.39.3.589. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kielian M. C., Keränen S., Käriäinen L., Helenius A. Membrane fusion mutants of Semliki Forest virus. J Cell Biol. 1984 Jan;98(1):139–145. doi: 10.1083/jcb.98.1.139. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kreis T. E., Lodish H. F. Oligomerization is essential for transport of vesicular stomatitis viral glycoprotein to the cell surface. Cell. 1986 Sep 12;46(6):929–937. doi: 10.1016/0092-8674(86)90075-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kreis T. E. Microinjected antibodies against the cytoplasmic domain of vesicular stomatitis virus glycoprotein block its transport to the cell surface. EMBO J. 1986 May;5(5):931–941. doi: 10.1002/j.1460-2075.1986.tb04306.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kvist S., Wiman K., Claesson L., Peterson P. A., Dobberstein B. Membrane insertion and oligomeric assembly of HLA-DR histocompatibility antigens. Cell. 1982 May;29(1):61–69. doi: 10.1016/0092-8674(82)90090-3. [DOI] [PubMed] [Google Scholar]
- Lafay F. Envelope proteins of vesicular stomatitis virus: effect of temperature-sensitive mutations in complementation groups III and V. J Virol. 1974 Nov;14(5):1220–1228. doi: 10.1128/jvi.14.5.1220-1228.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Li E., Tabas I., Kornfeld S. The synthesis of complex-type oligosaccharides. I. Structure of the lipid-linked oligosaccharide precursor of the complex-type oligosaccharides of the vesicular stomatitis virus G protein. J Biol Chem. 1978 Nov 10;253(21):7762–7770. [PubMed] [Google Scholar]
- Machamer C. E., Florkiewicz R. Z., Rose J. K. A single N-linked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus G protein to the cell surface. Mol Cell Biol. 1985 Nov;5(11):3074–3083. doi: 10.1128/mcb.5.11.3074. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mains P. E., Sibley C. H. The requirement of light chain for the surface deposition of the heavy chain of immunoglobulin M. J Biol Chem. 1983 Apr 25;258(8):5027–5033. [PubMed] [Google Scholar]
- Merlie J. P. Biogenesis of the acetylcholine receptor, a multisubunit integral membrane protein. Cell. 1984 Mar;36(3):573–575. doi: 10.1016/0092-8674(84)90335-0. [DOI] [PubMed] [Google Scholar]
- Merlie J. P., Lindstrom J. Assembly in vivo of mouse muscle acetylcholine receptor: identification of an alpha subunit species that may be an assembly intermediate. Cell. 1983 Oct;34(3):747–757. doi: 10.1016/0092-8674(83)90531-7. [DOI] [PubMed] [Google Scholar]
- Minami Y., Weissman A. M., Samelson L. E., Klausner R. D. Building a multichain receptor: synthesis, degradation, and assembly of the T-cell antigen receptor. Proc Natl Acad Sci U S A. 1987 May;84(9):2688–2692. doi: 10.1073/pnas.84.9.2688. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ronne H., Ocklind C., Wiman K., Rask L., Obrink B., Peterson P. A. Ligand-dependent regulation of intracellular protein transport: effect of vitamin a on the secretion of the retinol-binding protein. J Cell Biol. 1983 Mar;96(3):907–910. doi: 10.1083/jcb.96.3.907. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rose J. K., Bergmann J. E. Altered cytoplasmic domains affect intracellular transport of the vesicular stomatitis virus glycoprotein. Cell. 1983 Sep;34(2):513–524. doi: 10.1016/0092-8674(83)90384-7. [DOI] [PubMed] [Google Scholar]
- Rose J. K., Gallione C. J. Nucleotide sequences of the mRNA's encoding the vesicular stomatitis virus G and M proteins determined from cDNA clones containing the complete coding regions. J Virol. 1981 Aug;39(2):519–528. doi: 10.1128/jvi.39.2.519-528.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rose J. K., Welch W. J., Sefton B. M., Esch F. S., Ling N. C. Vesicular stomatitis virus glycoprotein is anchored in the viral membrane by a hydrophobic domain near the COOH terminus. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3884–3888. doi: 10.1073/pnas.77.7.3884. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rothman J. E., Lodish H. F. Synchronised transmembrane insertion and glycosylation of a nascent membrane protein. Nature. 1977 Oct 27;269(5631):775–780. doi: 10.1038/269775a0. [DOI] [PubMed] [Google Scholar]
- Saraste J., Kuismanen E. Pre- and post-Golgi vacuoles operate in the transport of Semliki Forest virus membrane glycoproteins to the cell surface. Cell. 1984 Sep;38(2):535–549. doi: 10.1016/0092-8674(84)90508-7. [DOI] [PubMed] [Google Scholar]
- Schnitzer T. J., Dickson C., Weiss R. A. Morphological and biochemical characterization of viral particles produced by the tsO45 mutant of vesicular stomatitis virus at restrictive temperature. J Virol. 1979 Jan;29(1):185–195. doi: 10.1128/jvi.29.1.185-195.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Severinsson L., Peterson P. A. Beta 2-microglobulin induces intracellular transport of human class I transplantation antigen heavy chains in Xenopus laevis oocytes. J Cell Biol. 1984 Jul;99(1 Pt 1):226–232. doi: 10.1083/jcb.99.1.226. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Silverberg M., Marchesi V. T. The anomalous electrophoretic behavior of the major sialoglycoprotein from the human erythrocyte. J Biol Chem. 1978 Jan 10;253(1):95–98. [PubMed] [Google Scholar]
- Tkacz J. S., Lampen O. Tunicamycin inhibition of polyisoprenyl N-acetylglucosaminyl pyrophosphate formation in calf-liver microsomes. Biochem Biophys Res Commun. 1975 Jul 8;65(1):248–257. doi: 10.1016/s0006-291x(75)80086-6. [DOI] [PubMed] [Google Scholar]
- Vogel R. H., Provencher S. W., von Bonsdorff C. H., Adrian M., Dubochet J. Envelope structure of Semliki Forest virus reconstructed from cryo-electron micrographs. Nature. 1986 Apr 10;320(6062):533–535. doi: 10.1038/320533a0. [DOI] [PubMed] [Google Scholar]
- White J., Kielian M., Helenius A. Membrane fusion proteins of enveloped animal viruses. Q Rev Biophys. 1983 May;16(2):151–195. doi: 10.1017/s0033583500005072. [DOI] [PubMed] [Google Scholar]
- White J., Matlin K., Helenius A. Cell fusion by Semliki Forest, influenza, and vesicular stomatitis viruses. J Cell Biol. 1981 Jun;89(3):674–679. doi: 10.1083/jcb.89.3.674. [DOI] [PMC free article] [PubMed] [Google Scholar]