Abstract
The vesicular stomatitis virus glycoprotein forms noncovalently linked trimers in the endoplasmic reticulum before being transported to the Golgi apparatus. The experiments reported here were designed to determine if the extracellular domain of the glycoprotein contains structural information sufficient to direct trimer formation. To accomplish this, we generated a construct encoding G protein with the normal transmembrane and anchor sequences replaced with the sequence encoding 53 C-terminal amino acids from the Thy-1.1 glycoprotein. We show here that these sequences were able to specify glycolipid addition to the truncated G protein, probably after cleavage of 31 amino acids derived from Thy-1.1. The glycolipid-anchored G protein formed trimers and was expressed on the cell surface in a form that could be cleaved by phosphoinositol-specific phospholipase C. However, the rate of transport was reduced, compared with that of wild-type G protein. A second form of the G protein was generated by deletion of only the transmembrane and cytoplasmic domains. This mutant protein also formed trimers with relatively high efficiency and was secreted slowly from cells.
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Selected References
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- Bangs J. D., Hereld D., Krakow J. L., Hart G. W., Englund P. T. Rapid processing of the carboxyl terminus of a trypanosome variant surface glycoprotein. Proc Natl Acad Sci U S A. 1985 May;82(10):3207–3211. doi: 10.1073/pnas.82.10.3207. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Caras I. W., Weddell G. N., Davitz M. A., Nussenzweig V., Martin D. W., Jr Signal for attachment of a phospholipid membrane anchor in decay accelerating factor. Science. 1987 Nov 27;238(4831):1280–1283. doi: 10.1126/science.2446389. [DOI] [PubMed] [Google Scholar]
- Conzelmann A., Spiazzi A., Bron C. Glycolipid anchors are attached to Thy-1 glycoprotein rapidly after translation. Biochem J. 1987 Sep 15;246(3):605–610. doi: 10.1042/bj2460605. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Doms R. W., Keller D. S., Helenius A., Balch W. E. Role for adenosine triphosphate in regulating the assembly and transport of vesicular stomatitis virus G protein trimers. J Cell Biol. 1987 Nov;105(5):1957–1969. doi: 10.1083/jcb.105.5.1957. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Doms R. W., Ruusala A., Machamer C., Helenius J., Helenius A., Rose J. K. Differential effects of mutations in three domains on folding, quaternary structure, and intracellular transport of vesicular stomatitis virus G protein. J Cell Biol. 1988 Jul;107(1):89–99. doi: 10.1083/jcb.107.1.89. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dunphy W. G., Rothman J. E. Compartmental organization of the Golgi stack. Cell. 1985 Aug;42(1):13–21. doi: 10.1016/s0092-8674(85)80097-0. [DOI] [PubMed] [Google Scholar]
- Fatemi S. H., Haas R., Jentoft N., Rosenberry T. L., Tartakoff A. M. The glycophospholipid anchor of Thy-1. Biosynthetic labeling experiments with wild-type and class E Thy-1 negative lymphomas. J Biol Chem. 1987 Apr 5;262(10):4728–4732. [PubMed] [Google Scholar]
- Felgner P. L., Gadek T. R., Holm M., Roman R., Chan H. W., Wenz M., Northrop J. P., Ringold G. M., Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7413–7417. doi: 10.1073/pnas.84.21.7413. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ferguson M. A., Williams A. F. Cell-surface anchoring of proteins via glycosyl-phosphatidylinositol structures. Annu Rev Biochem. 1988;57:285–320. doi: 10.1146/annurev.bi.57.070188.001441. [DOI] [PubMed] [Google Scholar]
- Florkiewicz R. Z., Rose J. K. A cell line expressing vesicular stomatitis virus glycoprotein fuses at low pH. Science. 1984 Aug 17;225(4663):721–723. doi: 10.1126/science.6087454. [DOI] [PubMed] [Google Scholar]
- Florkiewicz R. Z., Smith A., Bergmann J. E., Rose J. K. Isolation of stable mouse cell lines that express cell surface and secreted forms of the vesicular stomatitis virus glycoprotein. J Cell Biol. 1983 Nov;97(5 Pt 1):1381–1388. doi: 10.1083/jcb.97.5.1381. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fuerst T. R., Niles E. G., Studier F. W., Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122–8126. doi: 10.1073/pnas.83.21.8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gething M. J., Sambrook J. Construction of influenza haemagglutinin genes that code for intracellular and secreted forms of the protein. Nature. 1982 Dec 16;300(5893):598–603. doi: 10.1038/300598a0. [DOI] [PubMed] [Google Scholar]
- Giguére V., Isobe K., Grosveld F. Structure of the murine Thy-1 gene. EMBO J. 1985 Aug;4(8):2017–2024. doi: 10.1002/j.1460-2075.1985.tb03886.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Homans S. W., Ferguson M. A., Dwek R. A., Rademacher T. W., Anand R., Williams A. F. Complete structure of the glycosyl phosphatidylinositol membrane anchor of rat brain Thy-1 glycoprotein. Nature. 1988 May 19;333(6170):269–272. doi: 10.1038/333269a0. [DOI] [PubMed] [Google Scholar]
- Kornfeld R., Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. doi: 10.1146/annurev.bi.54.070185.003215. [DOI] [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]
- Low M. G., Kincade P. W. Phosphatidylinositol is the membrane-anchoring domain of the Thy-1 glycoprotein. Nature. 1985 Nov 7;318(6041):62–64. doi: 10.1038/318062a0. [DOI] [PubMed] [Google Scholar]
- Machamer C. E., Rose J. K. Vesicular stomatitis virus G proteins with altered glycosylation sites display temperature-sensitive intracellular transport and are subject to aberrant intermolecular disulfide bonding. J Biol Chem. 1988 Apr 25;263(12):5955–5960. [PubMed] [Google Scholar]
- Moore H. P., Kelly R. B. Secretory protein targeting in a pituitary cell line: differential transport of foreign secretory proteins to distinct secretory pathways. J Cell Biol. 1985 Nov;101(5 Pt 1):1773–1781. doi: 10.1083/jcb.101.5.1773. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Puddington L., Machamer C. E., Rose J. K. Cytoplasmic domains of cellular and viral integral membrane proteins substitute for the cytoplasmic domain of the vesicular stomatitis virus glycoprotein in transport to the plasma membrane. J Cell Biol. 1986 Jun;102(6):2147–2157. doi: 10.1083/jcb.102.6.2147. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Riedel H., Kondor-Koch C., Garoff H. Cell surface expression of fusogenic vesicular stomatitis virus G protein from cloned cDNA. EMBO J. 1984 Jul;3(7):1477–1483. doi: 10.1002/j.1460-2075.1984.tb01999.x. [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., Bergmann J. E. Expression from cloned cDNA of cell-surface secreted forms of the glycoprotein of vesicular stomatitis virus in eucaryotic cells. Cell. 1982 Oct;30(3):753–762. doi: 10.1016/0092-8674(82)90280-x. [DOI] [PubMed] [Google Scholar]
- Rose J. K., Doms R. W. Regulation of protein export from the endoplasmic reticulum. Annu Rev Cell Biol. 1988;4:257–288. doi: 10.1146/annurev.cb.04.110188.001353. [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]
- 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]
- Tarentino A. L., Maley F. Purification and properties of an endo-beta-N-acetylglucosaminidase from Streptomyces griseus. J Biol Chem. 1974 Feb 10;249(3):811–817. [PubMed] [Google Scholar]
- Tse A. G., Barclay A. N., Watts A., Williams A. F. A glycophospholipid tail at the carboxyl terminus of the Thy-1 glycoprotein of neurons and thymocytes. Science. 1985 Nov 29;230(4729):1003–1008. doi: 10.1126/science.2865810. [DOI] [PubMed] [Google Scholar]
- Tykocinski M. L., Shu H. K., Ayers D. J., Walter E. I., Getty R. R., Groger R. K., Hauer C. A., Medof M. E. Glycolipid reanchoring of T-lymphocyte surface antigen CD8 using the 3' end sequence of decay-accelerating factor's mRNA. Proc Natl Acad Sci U S A. 1988 May;85(10):3555–3559. doi: 10.1073/pnas.85.10.3555. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Whitt M. A., Chong L., Rose J. K. Glycoprotein cytoplasmic domain sequences required for rescue of a vesicular stomatitis virus glycoprotein mutant. J Virol. 1989 Sep;63(9):3569–3578. doi: 10.1128/jvi.63.9.3569-3578.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wieland F. T., Gleason M. L., Serafini T. A., Rothman J. E. The rate of bulk flow from the endoplasmic reticulum to the cell surface. Cell. 1987 Jul 17;50(2):289–300. doi: 10.1016/0092-8674(87)90224-8. [DOI] [PubMed] [Google Scholar]
- Wilson I. A., Skehel J. J., Wiley D. C. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature. 1981 Jan 29;289(5796):366–373. doi: 10.1038/289366a0. [DOI] [PubMed] [Google Scholar]