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. 1986 Dec 1;103(6):2607–2618. doi: 10.1083/jcb.103.6.2607

Alteration of the cytoplasmic domain of the membrane-spanning glycoprotein p62 of Semliki Forest virus does not affect its polar distribution in established lines of Madin-Darby canine kidney cells

PMCID: PMC2114582  PMID: 3539942

Abstract

Expression of the Semliki Forest virus p62/E2 protein was studied in the polarized epithelial cell line Madin-Darby canine kidney (MDCK). After infection this transmembrane protein, together with the other spike subunit E1, accumulates at the basolateral surface of MDCK cells (Fuller, S. D., C.-H. von Bonsdorff, and K. Simons, 1985, EMBO (Eur. Mol. Biol. Organ.) J., 4:2475-2485). The cDNAs encoding truncated forms of the protein were used to stably transform MDCK cells to examine the role of subunit oligomerization (E1-E2) and the cytoplasmic domain of p62/E2 in directed transport to the basolateral surface. The biochemical characteristics and polarity of the expressed proteins were studied using cell monolayers grown on nitrocellulose filters. A wild- type form of p62/E2, in the absence of E1, and two forms having either 15 or 3 of the wild-type 31-amino acid carboxyl cytoplasmic domain were all localized to the basolateral surface. These results indicate that the cytoplasmic domain of E2 does not contain the information essential for directed transport to the plasma membrane, and imply that this information resides in either the lumenal and/or membrane-spanning segments of this transmembrane protein.

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Selected References

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  1. Aliperti G., Schlesinger M. J. Evidence for an autoprotease activity of sindbis virus capsid protein. Virology. 1978 Oct 15;90(2):366–369. doi: 10.1016/0042-6822(78)90321-5. [DOI] [PubMed] [Google Scholar]
  2. Ash J. F., Louvard D., Singer S. J. Antibody-induced linkages of plasma membrane proteins to intracellular actomyosin-containing filaments in cultured fibroblasts. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5584–5588. doi: 10.1073/pnas.74.12.5584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Balcarova-Ständer J., Pfeiffer S. E., Fuller S. D., Simons K. Development of cell surface polarity in the epithelial Madin-Darby canine kidney (MDCK) cell line. EMBO J. 1984 Nov;3(11):2687–2694. doi: 10.1002/j.1460-2075.1984.tb02194.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berger M., Schmidt M. F. Protein fatty acyltransferase is located in the rough endoplasmic reticulum. FEBS Lett. 1985 Aug 5;187(2):289–294. doi: 10.1016/0014-5793(85)81261-8. [DOI] [PubMed] [Google Scholar]
  5. Blobel G., Dobberstein B. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol. 1975 Dec;67(3):835–851. doi: 10.1083/jcb.67.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blobel G., Dobberstein B. Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. J Cell Biol. 1975 Dec;67(3):852–862. doi: 10.1083/jcb.67.3.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bolivar F., Backman K. Plasmids of Escherichia coli as cloning vectors. Methods Enzymol. 1979;68:245–267. doi: 10.1016/0076-6879(79)68018-7. [DOI] [PubMed] [Google Scholar]
  8. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
  9. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  10. Bravo R., Fey S. J., Larsen P. M., Coppard N., Celis J. E. Proteins IEF (isoelectric focusing) 31 and IEF 46 are keratin-type components of the intermediate-sized filaments: keratins of various human cultured epithelial cells. J Cell Biol. 1983 Feb;96(2):416–423. doi: 10.1083/jcb.96.2.416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Burgess T. L., Craik C. S., Kelly R. B. The exocrine protein trypsinogen is targeted into the secretory granules of an endocrine cell line: studies by gene transfer. J Cell Biol. 1985 Aug;101(2):639–645. doi: 10.1083/jcb.101.2.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cereijido M., Robbins E. S., Dolan W. J., Rotunno C. A., Sabatini D. D. Polarized monolayers formed by epithelial cells on a permeable and translucent support. J Cell Biol. 1978 Jun;77(3):853–880. doi: 10.1083/jcb.77.3.853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chambard M., Gabrion J., Mauchamp J. Influence of collagen gel on the orientation of epithelial cell polarity: follicle formation from isolated thyroid cells and from preformed monolayers. J Cell Biol. 1981 Oct;91(1):157–166. doi: 10.1083/jcb.91.1.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cutler D. F., Garoff H. Mutants of the membrane-binding region of Semliki Forest virus E2 protein. I. Cell surface transport and fusogenic activity. J Cell Biol. 1986 Mar;102(3):889–901. doi: 10.1083/jcb.102.3.889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Cutler D. F., Melancon P., Garoff H. Mutants of the membrane-binding region of Semliki Forest virus E2 protein. II. Topology and membrane binding. J Cell Biol. 1986 Mar;102(3):902–910. doi: 10.1083/jcb.102.3.902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Davis N. G., Model P. An artificial anchor domain: hydrophobicity suffices to stop transfer. Cell. 1985 Jun;41(2):607–614. doi: 10.1016/s0092-8674(85)80033-7. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Fujiki Y., Hubbard A. L., Fowler S., Lazarow P. B. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol. 1982 Apr;93(1):97–102. doi: 10.1083/jcb.93.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fuller S. D., von Bonsdorff C. H., Simons K. Cell surface influenza haemagglutinin can mediate infection by other animal viruses. EMBO J. 1985 Oct;4(10):2475–2485. doi: 10.1002/j.1460-2075.1985.tb03959.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fuller S., von Bonsdorff C. H., Simons K. Vesicular stomatitis virus infects and matures only through the basolateral surface of the polarized epithelial cell line, MDCK. Cell. 1984 Aug;38(1):65–77. doi: 10.1016/0092-8674(84)90527-0. [DOI] [PubMed] [Google Scholar]
  22. GREENWOOD F. C., HUNTER W. M., GLOVER J. S. THE PREPARATION OF I-131-LABELLED HUMAN GROWTH HORMONE OF HIGH SPECIFIC RADIOACTIVITY. Biochem J. 1963 Oct;89:114–123. doi: 10.1042/bj0890114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Garoff H., Kondor-Koch C., Riedel H. Structure and assembly of alphaviruses. Curr Top Microbiol Immunol. 1982;99:1–50. doi: 10.1007/978-3-642-68528-6_1. [DOI] [PubMed] [Google Scholar]
  25. Garoff H., Simons K., Dobberstein B. Assembly of the Semliki Forest virus membrane glycoproteins in the membrane of the endoplasmic reticulum in vitro. J Mol Biol. 1978 Oct 5;124(4):587–600. doi: 10.1016/0022-2836(78)90173-0. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Green R. F., Meiss H. K., Rodriguez-Boulan E. Glycosylation does not determine segregation of viral envelope proteins in the plasma membrane of epithelial cells. J Cell Biol. 1981 May;89(2):230–239. doi: 10.1083/jcb.89.2.230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  29. Kondor-Koch C., Bravo R., Fuller S. D., Cutler D., Garoff H. Exocytotic pathways exist to both the apical and the basolateral cell surface of the polarized epithelial cell MDCK. Cell. 1985 Nov;43(1):297–306. doi: 10.1016/0092-8674(85)90035-2. [DOI] [PubMed] [Google Scholar]
  30. Kondor-Koch C., Burke B., Garoff H. Expression of Semliki Forest virus proteins from cloned complementary DNA. I. The fusion activity of the spike glycoprotein. J Cell Biol. 1983 Sep;97(3):644–651. doi: 10.1083/jcb.97.3.644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Käriäinen L., Simons K., von Bonsdorff C. H. Studies in subviral components of Semliki Forest virus. Ann Med Exp Biol Fenn. 1969;47(4):235–248. [PubMed] [Google Scholar]
  33. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  34. 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]
  35. Louvard D. Apical membrane aminopeptidase appears at site of cell-cell contact in cultured kidney epithelial cells. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4132–4136. doi: 10.1073/pnas.77.7.4132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Louvard D., Reggio H., Warren G. Antibodies to the Golgi complex and the rough endoplasmic reticulum. J Cell Biol. 1982 Jan;92(1):92–107. doi: 10.1083/jcb.92.1.92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Matlin K. S., Simons K. Reduced temperature prevents transfer of a membrane glycoprotein to the cell surface but does not prevent terminal glycosylation. Cell. 1983 Aug;34(1):233–243. doi: 10.1016/0092-8674(83)90154-x. [DOI] [PubMed] [Google Scholar]
  38. Meyer D. I., Dobberstein B. Identification and characterization of a membrane component essential for the translocation of nascent proteins across the membrane of the endoplasmic reticulum. J Cell Biol. 1980 Nov;87(2 Pt 1):503–508. doi: 10.1083/jcb.87.2.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Misek D. E., Bard E., Rodriguez-Boulan E. Biogenesis of epithelial cell polarity: intracellular sorting and vectorial exocytosis of an apical plasma membrane glycoprotein. Cell. 1984 Dec;39(3 Pt 2):537–546. doi: 10.1016/0092-8674(84)90460-4. [DOI] [PubMed] [Google Scholar]
  40. Moore H. P., Walker M. D., Lee F., Kelly R. B. Expressing a human proinsulin cDNA in a mouse ACTH-secreting cell. Intracellular storage, proteolytic processing, and secretion on stimulation. Cell. 1983 Dec;35(2 Pt 1):531–538. doi: 10.1016/0092-8674(83)90187-3. [DOI] [PubMed] [Google Scholar]
  41. Perkins F. M., Handler J. S. Transport properties of toad kidney epithelia in culture. Am J Physiol. 1981 Sep;241(3):C154–C159. doi: 10.1152/ajpcell.1981.241.3.C154. [DOI] [PubMed] [Google Scholar]
  42. Pfeiffer S., Fuller S. D., Simons K. Intracellular sorting and basolateral appearance of the G protein of vesicular stomatitis virus in Madin-Darby canine kidney cells. J Cell Biol. 1985 Aug;101(2):470–476. doi: 10.1083/jcb.101.2.470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Poruchynsky M. S., Tyndall C., Both G. W., Sato F., Bellamy A. R., Atkinson P. H. Deletions into an NH2-terminal hydrophobic domain result in secretion of rotavirus VP7, a resident endoplasmic reticulum membrane glycoprotein. J Cell Biol. 1985 Dec;101(6):2199–2209. doi: 10.1083/jcb.101.6.2199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Richardson J. C., Scalera V., Simmons N. L. Identification of two strains of MDCK cells which resemble separate nephron tubule segments. Biochim Biophys Acta. 1981 Feb 18;673(1):26–36. [PubMed] [Google Scholar]
  45. Rindler M. J., Chuman L. M., Shaffer L., Saier M. H., Jr Retention of differentiated properties in an established dog kidney epithelial cell line (MDCK). J Cell Biol. 1979 Jun;81(3):635–648. doi: 10.1083/jcb.81.3.635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rindler M. J., Ivanov I. E., Plesken H., Sabatini D. D. Polarized delivery of viral glycoproteins to the apical and basolateral plasma membranes of Madin-Darby canine kidney cells infected with temperature-sensitive viruses. J Cell Biol. 1985 Jan;100(1):136–151. doi: 10.1083/jcb.100.1.136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Rodriguez Boulan E., Sabatini D. D. Asymmetric budding of viruses in epithelial monlayers: a model system for study of epithelial polarity. Proc Natl Acad Sci U S A. 1978 Oct;75(10):5071–5075. doi: 10.1073/pnas.75.10.5071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Roman L. M., Hubbard A. L. A domain-specific marker for the hepatocyte plasma membrane. III. Isolation of bile canalicular membrane by immunoadsorption. J Cell Biol. 1984 Apr;98(4):1497–1504. doi: 10.1083/jcb.98.4.1497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Roman L. M., Hubbard A. L. A domain-specific marker for the hepatocyte plasma membrane: localization of leucine aminopeptidase to the bile canalicular domain. J Cell Biol. 1983 Jun;96(6):1548–1558. doi: 10.1083/jcb.96.6.1548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. 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]
  52. Roth M. G., Compans R. W., Giusti L., Davis A. R., Nayak D. P., Gething M. J., Sambrook J. Influenza virus hemagglutinin expression is polarized in cells infected with recombinant SV40 viruses carrying cloned hemagglutinin DNA. Cell. 1983 Jun;33(2):435–443. doi: 10.1016/0092-8674(83)90425-7. [DOI] [PubMed] [Google Scholar]
  53. Roth M. G., Fitzpatrick J. P., Compans R. W. Polarity of influenza and vesicular stomatitis virus maturation in MDCK cells: lack of a requirement for glycosylation of viral glycoproteins. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6430–6434. doi: 10.1073/pnas.76.12.6430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Rüther U., Wagner E. F., Müller R. Analysis of the differentiation-promoting potential of inducible c-fos genes introduced into embryonal carcinoma cells. EMBO J. 1985 Jul;4(7):1775–1781. doi: 10.1002/j.1460-2075.1985.tb03850.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. 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]
  56. Simons K., Fuller S. D. Cell surface polarity in epithelia. Annu Rev Cell Biol. 1985;1:243–288. doi: 10.1146/annurev.cb.01.110185.001331. [DOI] [PubMed] [Google Scholar]
  57. Simons K., Warren G. Semliki Forest virus: a probe for membrane traffic in the animal cell. Adv Protein Chem. 1984;36:79–132. doi: 10.1016/S0065-3233(08)60296-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Steck T. L., Yu J. Selective solubilization of proteins from red blood cell membranes by protein perturbants. J Supramol Struct. 1973;1(3):220–232. doi: 10.1002/jss.400010307. [DOI] [PubMed] [Google Scholar]
  59. Stephens E. B., Compans R. W., Earl P., Moss B. Surface expression of viral glycoproteins is polarized in epithelial cells infected with recombinant vaccinia viral vectors. EMBO J. 1986 Feb;5(2):237–245. doi: 10.1002/j.1460-2075.1986.tb04204.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Suissa M. Spectrophotometric quantitation of silver grains eluted from autoradiograms. Anal Biochem. 1983 Sep;133(2):511–514. doi: 10.1016/0003-2697(83)90117-3. [DOI] [PubMed] [Google Scholar]
  61. 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]
  62. Walter P., Blobel G. Purification of a membrane-associated protein complex required for protein translocation across the endoplasmic reticulum. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7112–7116. doi: 10.1073/pnas.77.12.7112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Ziemiecki A., Garoff H., Simons K. Formation of the Semliki Forest virus membrane glycoprotein complexes in the infected cell. J Gen Virol. 1980 Sep;50(1):111–123. doi: 10.1099/0022-1317-50-1-111. [DOI] [PubMed] [Google Scholar]
  64. 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]
  65. 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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