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. 1986 Jun 1;102(6):2147–2157. doi: 10.1083/jcb.102.6.2147

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

PMCID: PMC2114239  PMID: 3011809

Abstract

Oligonucleotide-directed mutagenesis was used to construct chimeric cDNAs that encode the extracellular and transmembrane domains of the vesicular stomatitis virus glycoprotein (G) linked to the cytoplasmic domain of either the immunoglobulin mu membrane heavy chain, the hemagglutinin glycoprotein of influenza virus, or the small glycoprotein (p23) of infectious bronchitis virus. Biochemical analyses and immunofluorescence microscopy demonstrated that these hybrid genes were correctly expressed in eukaryotic cells and that the hybrid proteins were transported to the plasma membrane. The rate of transport to the Golgi complex of G protein with an immunoglobulin mu membrane cytoplasmic domain was approximately sixfold slower than G protein with its normal cytoplasmic domain. However, this rate was virtually identical to the rate of transport of micron heavy chain molecules measured in the B cell line WEHI 231. The rate of transport of G protein with a hemagglutinin cytoplasmic domain was threefold slower than wild type G protein and G protein with a p23 cytoplasmic domain, which were transported at similar rates. The combined results underscore the importance of the amino acid sequence in the cytoplasmic domain for efficient transport of G protein to the cell surface. Also, normal cytoplasmic domains from other transmembrane glycoproteins can substitute for the G protein cytoplasmic domain in transport of G protein to the plasma membrane. The method of constructing precise hybrid proteins described here will be useful in defining functions of specific domains of viral and cellular integral membrane proteins.

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Selected References

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  1. Adams G. A., Rose J. K. Incorporation of a charged amino acid into the membrane-spanning domain blocks cell surface transport but not membrane anchoring of a viral glycoprotein. Mol Cell Biol. 1985 Jun;5(6):1442–1448. doi: 10.1128/mcb.5.6.1442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adams G. A., Rose J. K. Structural requirements of a membrane-spanning domain for protein anchoring and cell surface transport. Cell. 1985 Jul;41(3):1007–1015. doi: 10.1016/s0092-8674(85)80081-7. [DOI] [PubMed] [Google Scholar]
  3. Argon Y., Milstein C. Intracellular processing of membrane and secreted immunoglobulin delta-chains. J Immunol. 1984 Sep;133(3):1627–1634. [PubMed] [Google Scholar]
  4. Arnheiter H., Dubois-Dalcq M., Lazzarini R. A. Direct visualization of protein transport and processing in the living cell by microinjection of specific antibodies. Cell. 1984 Nov;39(1):99–109. doi: 10.1016/0092-8674(84)90195-8. [DOI] [PubMed] [Google Scholar]
  5. Beale D., Feinstein A. Studies on the reduction of a human 19S immunoglobulin M. Biochem J. 1969 Apr;112(2):187–194. doi: 10.1042/bj1120187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blattner F. R., Tucker P. W. The molecular biology of immunoglobulin D. Nature. 1984 Feb 2;307(5950):417–422. doi: 10.1038/307417a0. [DOI] [PubMed] [Google Scholar]
  7. Blobel G. Intracellular protein topogenesis. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1496–1500. doi: 10.1073/pnas.77.3.1496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  9. Boursnell M. E., Brown T. D., Binns M. M. Sequence of the membrane protein gene from avian coronavirus IBV. Virus Res. 1984;1(4):303–313. doi: 10.1016/0168-1702(84)90019-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Brown W. J., Constantinescu E., Farquhar M. G. Redistribution of mannose-6-phosphate receptors induced by tunicamycin and chloroquine. J Cell Biol. 1984 Jul;99(1 Pt 1):320–326. doi: 10.1083/jcb.99.1.320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cheng H. L., Blattner F. R., Fitzmaurice L., Mushinski J. F., Tucker P. W. Structure of genes for membrane and secreted murine IgD heavy chains. Nature. 1982 Apr 1;296(5856):410–415. doi: 10.1038/296410a0. [DOI] [PubMed] [Google Scholar]
  12. Creager R. S., Cardamone J. J., Jr, Youngner J. S. Human lymphoblastoid cell lines of B- and T-cell origin: different responses to infection with vesicular stomatitis virus. Virology. 1981 May;111(1):211–222. doi: 10.1016/0042-6822(81)90666-8. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Dubois-Dalcq M. E., Doller E. W., Haspel M. V., Holmes K. V. Cell tropism and expression of mouse hepatitis viruses (MHV) in mouse spinal cord cultures. Virology. 1982 Jun;119(2):317–331. doi: 10.1016/0042-6822(82)90092-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dulis B. H., Kloppel T. M., Grey H. M., Kubo R. T. Regulation of catabolism of IgM heavy chains in a B lymphoma cell line. J Biol Chem. 1982 Apr 25;257(8):4369–4374. [PubMed] [Google Scholar]
  17. Garoff H., Kondor-Koch C., Pettersson R., Burke B. Expression of Semliki Forest virus proteins from cloned complementary DNA. II. The membrane-spanning glycoprotein E2 is transported to the cell surface without its normal cytoplasmic domain. J Cell Biol. 1983 Sep;97(3):652–658. doi: 10.1083/jcb.97.3.652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  20. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gumbiner B., Kelly R. B. Two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells. Cell. 1982 Jan;28(1):51–59. doi: 10.1016/0092-8674(82)90374-9. [DOI] [PubMed] [Google Scholar]
  22. Hubbard S. C., Ivatt R. J. Synthesis and processing of asparagine-linked oligosaccharides. Annu Rev Biochem. 1981;50:555–583. doi: 10.1146/annurev.bi.50.070181.003011. [DOI] [PubMed] [Google Scholar]
  23. Katz F. N., Rothman J. E., Lingappa V. R., Blobel G., Lodish H. F. Membrane assembly in vitro: synthesis, glycosylation, and asymmetric insertion of a transmembrane protein. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3278–3282. doi: 10.1073/pnas.74.8.3278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kehry M., Ewald S., Douglas R., Sibley C., Raschke W., Fambrough D., Hood L. The immunoglobulin mu chains of membrane-bound and secreted IgM molecules differ in their C-terminal segments. Cell. 1980 Sep;21(2):393–406. doi: 10.1016/0092-8674(80)90476-6. [DOI] [PubMed] [Google Scholar]
  25. Knipe D. M., Baltimore D., Lodish H. F. Maturation of viral proteins in cells infected with temperature-sensitive mutants of vesicular stomatitis virus. J Virol. 1977 Mar;21(3):1149–1158. doi: 10.1128/jvi.21.3.1149-1158.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  27. Lamson G., Koshland M. E. Changes in J chain and mu chain RNA expression as a function of B cell differentiation. J Exp Med. 1984 Sep 1;160(3):877–892. doi: 10.1084/jem.160.3.877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  29. Laskov R., Kim J. K., Woods V. L., McKeever P. E., Asofsky R. Membrane immunoglobulins of spontaneous B lymphomas of aged BALB/c mice. Eur J Immunol. 1981 Jun;11(6):462–468. doi: 10.1002/eji.1830110605. [DOI] [PubMed] [Google Scholar]
  30. Lehrman M. A., Goldstein J. L., Brown M. S., Russell D. W., Schneider W. J. Internalization-defective LDL receptors produced by genes with nonsense and frameshift mutations that truncate the cytoplasmic domain. Cell. 1985 Jul;41(3):735–743. doi: 10.1016/s0092-8674(85)80054-4. [DOI] [PubMed] [Google Scholar]
  31. Lyles D. S., McConnell K. A. Subcellular localization of the env-related glycoproteins in Friend erythroleukemia cells. J Virol. 1981 Jul;39(1):263–272. doi: 10.1128/jvi.39.1.263-272.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Mather E. L., Nelson K. J., Haimovich J., Perry R. P. Mode of regulation of immunoglobulin mu- and delta-chain expression varies during B-lymphocyte maturation. Cell. 1984 Feb;36(2):329–338. doi: 10.1016/0092-8674(84)90226-5. [DOI] [PubMed] [Google Scholar]
  34. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. McCune J. M., Lingappa V. R., Fu S. M., Blobel G., Kunkel H. G. Biogenesis of membrane-bound and secreted immunoglobulins. I. Two distinct translation products of human mu-chain, with identical N-termini and different C-termini. J Exp Med. 1980 Aug 1;152(2):463–468. doi: 10.1084/jem.152.2.463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McQueen N. L., Nayak D. P., Jones L. V., Compans R. W. Chimeric influenza virus hemagglutinin containing either the NH2 terminus or the COOH terminus of G protein of vesicular stomatitis virus is defective in transport to the cell surface. Proc Natl Acad Sci U S A. 1984 Jan;81(2):395–399. doi: 10.1073/pnas.81.2.395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Morrison T. G. Site of synthesis of membrane and nonmembrane proteins of vesicular stomatitis virus. J Biol Chem. 1975 Sep 10;250(17):6955–6962. [PubMed] [Google Scholar]
  38. Murre C., Reiss C. S., Bernabeu C., Chen L. B., Burakoff S. J., Seidman J. G. Construction, expression and recognition of an H-2 molecule lacking its carboxyl terminus. Nature. 1984 Feb 2;307(5950):432–436. doi: 10.1038/307432a0. [DOI] [PubMed] [Google Scholar]
  39. Niemann H., Boschek B., Evans D., Rosing M., Tamura T., Klenk H. D. Post-translational glycosylation of coronavirus glycoprotein E1: inhibition by monensin. EMBO J. 1982;1(12):1499–1504. doi: 10.1002/j.1460-2075.1982.tb01346.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Puddington L., Lively M. O., Lyles D. S. Role of the nuclear envelope in synthesis, processing, and transport of membrane glycoproteins. J Biol Chem. 1985 May 10;260(9):5641–5647. [PubMed] [Google Scholar]
  41. Raschke W. C., Mather E. L., Koshland M. E. Assembly and secretion of pentameric IgM in a fusion between a nonsecreting B cell lymphoma and an IgG-secreting plasmacytoma. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3469–3473. doi: 10.1073/pnas.76.7.3469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Riedel H. Different membrane anchors allow the Semliki Forest virus spike subunit E2 to reach the cell surface. J Virol. 1985 Apr;54(1):224–228. doi: 10.1128/jvi.54.1.224-228.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rogers J., Early P., Carter C., Calame K., Bond M., Hood L., Wall R. Two mRNAs with different 3' ends encode membrane-bound and secreted forms of immunoglobulin mu chain. Cell. 1980 Jun;20(2):303–312. doi: 10.1016/0092-8674(80)90616-9. [DOI] [PubMed] [Google Scholar]
  44. Rose J. K., Adams G. A., Gallione C. J. The presence of cysteine in the cytoplasmic domain of the vesicular stomatitis virus glycoprotein is required for palmitate addition. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2050–2054. doi: 10.1073/pnas.81.7.2050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. 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]
  47. 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]
  48. 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]
  49. Rottier P. J., Welling G. W., Welling-Wester S., Niesters H. G., Lenstra J. A., Van der Zeijst B. A. Predicted membrane topology of the coronavirus protein E1. Biochemistry. 1986 Mar 25;25(6):1335–1339. doi: 10.1021/bi00354a022. [DOI] [PubMed] [Google Scholar]
  50. Sabatini D. D., Kreibich G., Morimoto T., Adesnik M. Mechanisms for the incorporation of proteins in membranes and organelles. J Cell Biol. 1982 Jan;92(1):1–22. doi: 10.1083/jcb.92.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sibley C. H., Ewald S. J., Kehry M. R., Douglas R. H., Raschke W. C., Hood L. E. Characterization of multiple immunoglobulin mu-chains synthesized by two clones of a B cell lymphoma. J Immunol. 1980 Nov;125(5):2097–2105. [PubMed] [Google Scholar]
  52. Sidman C. B lymphocyte differentiation and the control of IgM mu chain expression. Cell. 1981 Feb;23(2):379–389. doi: 10.1016/0092-8674(81)90133-1. [DOI] [PubMed] [Google Scholar]
  53. Sprague J., Condra J. H., Arnheiter H., Lazzarini R. A. Expression of a recombinant DNA gene coding for the vesicular stomatitis virus nucleocapsid protein. J Virol. 1983 Feb;45(2):773–781. doi: 10.1128/jvi.45.2.773-781.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Strous G. J., Lodish H. F. Intracellular transport of secretory and membrane proteins in hepatoma cells infected by vesicular stomatitis virus. Cell. 1980 Dec;22(3):709–717. doi: 10.1016/0092-8674(80)90547-4. [DOI] [PubMed] [Google Scholar]
  55. Sveda M. M., Markoff L. J., Lai C. J. Cell surface expression of the influenza virus hemagglutinin requires the hydrophobic carboxy-terminal sequences. Cell. 1982 Sep;30(2):649–656. doi: 10.1016/0092-8674(82)90261-6. [DOI] [PubMed] [Google Scholar]
  56. Tabas I., Kornfeld S. Purification and characterization of a rat liver Golgi alpha-mannosidase capable of processing asparagine-linked oligosaccharides. J Biol Chem. 1979 Nov 25;254(22):11655–11663. [PubMed] [Google Scholar]
  57. Tarentino A. L., Plummer T. H., Jr, Maley F. The release of intact oligosaccharides from specific glycoproteins by endo-beta-N-acetylglucosaminidase H. J Biol Chem. 1974 Feb 10;249(3):818–824. [PubMed] [Google Scholar]
  58. Tartakoff A., Vassalli P. Plasma cell immunoglobulin M molecules. Their biosynthesis, assembly, and intracellular transport. J Cell Biol. 1979 Nov;83(2 Pt 1):284–299. doi: 10.1083/jcb.83.2.284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Verhoeyen M., Fang R., Jou W. M., Devos R., Huylebroeck D., Saman E., Fiers W. Antigenic drift between the haemagglutinin of the Hong Kong influenza strains A/Aichi/2/68 and A/Victoria/3/75. Nature. 1980 Aug 21;286(5775):771–776. doi: 10.1038/286771a0. [DOI] [PubMed] [Google Scholar]
  60. Wills J. W., Srinivas R. V., Hunter E. Mutations of the Rous sarcoma virus env gene that affect the transport and subcellular location of the glycoprotein products. J Cell Biol. 1984 Dec;99(6):2011–2023. doi: 10.1083/jcb.99.6.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. 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]
  62. Word C. J., Kuehl W. M. Expression of surface and secreted IgG2a by a murine B-lymphoma before and after hybridization to myeloma cells. Mol Immunol. 1981 Apr;18(4):311–322. doi: 10.1016/0161-5890(81)90055-9. [DOI] [PubMed] [Google Scholar]
  63. Zuniga M. C., Hood L. E. Clonal variation in cell surface display of an H-2 protein lacking a cytoplasmic tail. J Cell Biol. 1986 Jan;102(1):1–10. doi: 10.1083/jcb.102.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Zuniga M. C., Malissen B., McMillan M., Brayton P. R., Clark S. S., Forman J., Hood L. Expression and function of transplantation antigens with altered or deleted cytoplasmic domains. Cell. 1983 Sep;34(2):535–544. doi: 10.1016/0092-8674(83)90386-0. [DOI] [PubMed] [Google Scholar]

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