Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1997 Jun;71(6):4717–4727. doi: 10.1128/jvi.71.6.4717-4727.1997

A retention signal necessary and sufficient for Golgi localization maps to the cytoplasmic tail of a Bunyaviridae (Uukuniemi virus) membrane glycoprotein.

A M Andersson 1, L Melin 1, A Bean 1, R F Pettersson 1
PMCID: PMC191693  PMID: 9151865

Abstract

Members of the Bunyaviridae family mature by a budding process in the Golgi complex. The site of maturation is thought to be largely determined by the accumulation of the two spike glycoproteins, G1 and G2, in this organelle. Here we show that the signal for localizing the Uukuniemi virus (a phlebovirus) spike protein complex to the Golgi complex resides in the cytoplasmic tail of G1. We constructed chimeric proteins in which the ectodomain, transmembrane domain (TMD), and cytoplasmic tail (CT) of Uukuniemi virus G1 were exchanged with the corresponding domains of either vesicular stomatitis virus G protein (VSV G), chicken lysozyme, or CD4, all proteins readily transported to the plasma membrane. The chimeras were expressed in HeLa or BHK-21 cells by using either the T7 RNA polymerase-driven vaccinia virus system or the Semliki Forest virus system. The fate of the chimeric proteins was monitored by indirect immunofluorescence, and their localizations were compared by double labeling with markers specific for the Golgi complex. The results showed that the ectodomain and TMD (including the 10 flanking residues on either side of the membrane) of G1 played no apparent role in targeting chimeric proteins to the Golgi complex. Instead, all chimeras containing the CT of G1 were efficiently targeted to the Golgi complex and colocalized with mannosidase II, a Golgi-specific enzyme. Conversely, replacing the CT of G1 with that from VSV G resulted in the efficient transport of the chimeric protein to the cell surface. Progressive deletions of the G1 tail suggested that the Golgi retention signal maps to a region encompassing approximately residues 10 to 50, counting from the proposed border between the TMD and the tail. Both G1 and G2 were found to be acylated, as shown by incorporation of [3H]palmitate into the viral proteins. By mutational analyses of CD4-G1 chimeras, the sites for palmitylation were mapped to two closely spaced cysteine residues in the G1 tail. Changing either or both of these cysteines to alanine had no effect on the targeting of the chimeric protein to the Golgi complex.

Full Text

The Full Text of this article is available as a PDF (1.8 MB).

Selected References

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

  1. Andersson A. M., Melin L., Persson R., Raschperger E., Wikström L., Pettersson R. F. Processing and membrane topology of the spike proteins G1 and G2 of Uukuniemi virus. J Virol. 1997 Jan;71(1):218–225. doi: 10.1128/jvi.71.1.218-225.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Armstrong J., Patel S. The Golgi sorting domain of coronavirus E1 protein. J Cell Sci. 1991 Apr;98(Pt 4):567–575. doi: 10.1242/jcs.98.4.567. [DOI] [PubMed] [Google Scholar]
  3. Bretscher M. S., Munro S. Cholesterol and the Golgi apparatus. Science. 1993 Sep 3;261(5126):1280–1281. doi: 10.1126/science.8362242. [DOI] [PubMed] [Google Scholar]
  4. Bupp K., Stillmock K., González-Scarano F. Analysis of the intracellular transport properties of recombinant La Crosse virus glycoproteins. Virology. 1996 Jun 15;220(2):485–490. doi: 10.1006/viro.1996.0336. [DOI] [PubMed] [Google Scholar]
  5. Burke J., Pettitt J. M., Schachter H., Sarkar M., Gleeson P. A. The transmembrane and flanking sequences of beta 1,2-N-acetylglucosaminyltransferase I specify medial-Golgi localization. J Biol Chem. 1992 Dec 5;267(34):24433–24440. [PubMed] [Google Scholar]
  6. Crise B., Rose J. K. Identification of palmitoylation sites on CD4, the human immunodeficiency virus receptor. J Biol Chem. 1992 Jul 5;267(19):13593–13597. [PubMed] [Google Scholar]
  7. Elliott R. M., Schmaljohn C. S., Collett M. S. Bunyaviridae genome structure and gene expression. Curr Top Microbiol Immunol. 1991;169:91–141. doi: 10.1007/978-3-642-76018-1_4. [DOI] [PubMed] [Google Scholar]
  8. Elroy-Stein O., Fuerst T. R., Moss B. Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5' sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6126–6130. doi: 10.1073/pnas.86.16.6126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Funke C., Becker S., Dartsch H., Klenk H. D., Mühlberger E. Acylation of the Marburg virus glycoprotein. Virology. 1995 Apr 1;208(1):289–297. doi: 10.1006/viro.1995.1151. [DOI] [PubMed] [Google Scholar]
  10. Hobman T. C., Woodward L., Farquhar M. G. Targeting of a heterodimeric membrane protein complex to the Golgi: rubella virus E2 glycoprotein contains a transmembrane Golgi retention signal. Mol Biol Cell. 1995 Jan;6(1):7–20. doi: 10.1091/mbc.6.1.7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ivanova L., Schlesinger M. J. Site-directed mutations in the Sindbis virus E2 glycoprotein identify palmitoylation sites and affect virus budding. J Virol. 1993 May;67(5):2546–2551. doi: 10.1128/jvi.67.5.2546-2551.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jasmin B. J., Cartaud J., Bornens M., Changeux J. P. Golgi apparatus in chick skeletal muscle: changes in its distribution during end plate development and after denervation. Proc Natl Acad Sci U S A. 1989 Sep;86(18):7218–7222. doi: 10.1073/pnas.86.18.7218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jin H., Subbarao K., Bagai S., Leser G. P., Murphy B. R., Lamb R. A. Palmitylation of the influenza virus hemagglutinin (H3) is not essential for virus assembly or infectivity. J Virol. 1996 Mar;70(3):1406–1414. doi: 10.1128/jvi.70.3.1406-1414.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Klumperman J., Locker J. K., Meijer A., Horzinek M. C., Geuze H. J., Rottier P. J. Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion budding. J Virol. 1994 Oct;68(10):6523–6534. doi: 10.1128/jvi.68.10.6523-6534.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kuismanen E., Bång B., Hurme M., Pettersson R. F. Uukuniemi virus maturation: immunofluorescence microscopy with monoclonal glycoprotein-specific antibodies. J Virol. 1984 Jul;51(1):137–146. doi: 10.1128/jvi.51.1.137-146.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kuismanen E., Hedman K., Saraste J., Pettersson R. F. Uukuniemi virus maturation: accumulation of virus particles and viral antigens in the Golgi complex. Mol Cell Biol. 1982 Nov;2(11):1444–1458. doi: 10.1128/mcb.2.11.1444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kuismanen E. Posttranslational processing of Uukuniemi virus glycoproteins G1 and G2. J Virol. 1984 Sep;51(3):806–812. doi: 10.1128/jvi.51.3.806-812.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kuismanen E., Saraste J., Pettersson R. F. Effect of monensin on the assembly of Uukuniemi virus in the Golgi complex. J Virol. 1985 Sep;55(3):813–822. doi: 10.1128/jvi.55.3.813-822.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lappin D. F., Nakitare G. W., Palfreyman J. W., Elliott R. M. Localization of Bunyamwera bunyavirus G1 glycoprotein to the Golgi requires association with G2 but not with NSm. J Gen Virol. 1994 Dec;75(Pt 12):3441–3451. doi: 10.1099/0022-1317-75-12-3441. [DOI] [PubMed] [Google Scholar]
  20. Liljeström P., Garoff H. A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology (N Y) 1991 Dec;9(12):1356–1361. doi: 10.1038/nbt1291-1356. [DOI] [PubMed] [Google Scholar]
  21. Locker J. K., Klumperman J., Oorschot V., Horzinek M. C., Geuze H. J., Rottier P. J. The cytoplasmic tail of mouse hepatitis virus M protein is essential but not sufficient for its retention in the Golgi complex. J Biol Chem. 1994 Nov 11;269(45):28263–28269. [PubMed] [Google Scholar]
  22. Machamer C. E., Rose J. K. A specific transmembrane domain of a coronavirus E1 glycoprotein is required for its retention in the Golgi region. J Cell Biol. 1987 Sep;105(3):1205–1214. doi: 10.1083/jcb.105.3.1205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Matsuoka Y., Chen S. Y., Compans R. W. A signal for Golgi retention in the bunyavirus G1 glycoprotein. J Biol Chem. 1994 Sep 9;269(36):22565–22573. [PubMed] [Google Scholar]
  24. Melin L., Persson R., Andersson A., Bergström A., Rönnholm R., Pettersson R. F. The membrane glycoprotein G1 of Uukuniemi virus contains a signal for localization to the Golgi complex. Virus Res. 1995 Apr;36(1):49–66. doi: 10.1016/0168-1702(95)00006-C. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Moremen K. W., Touster O., Robbins P. W. Novel purification of the catalytic domain of Golgi alpha-mannosidase II. Characterization and comparison with the intact enzyme. J Biol Chem. 1991 Sep 5;266(25):16876–16885. [PubMed] [Google Scholar]
  26. Munro S. An investigation of the role of transmembrane domains in Golgi protein retention. EMBO J. 1995 Oct 2;14(19):4695–4704. doi: 10.1002/j.1460-2075.1995.tb00151.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Munro S. Sequences within and adjacent to the transmembrane segment of alpha-2,6-sialyltransferase specify Golgi retention. EMBO J. 1991 Dec;10(12):3577–3588. doi: 10.1002/j.1460-2075.1991.tb04924.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Naeve C. W., Williams D. Fatty acids on the A/Japan/305/57 influenza virus hemagglutinin have a role in membrane fusion. EMBO J. 1990 Dec;9(12):3857–3866. doi: 10.1002/j.1460-2075.1990.tb07604.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Naim H. Y., Amarneh B., Ktistakis N. T., Roth M. G. Effects of altering palmitylation sites on biosynthesis and function of the influenza virus hemagglutinin. J Virol. 1992 Dec;66(12):7585–7588. doi: 10.1128/jvi.66.12.7585-7588.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nilsson T., Hoe M. H., Slusarewicz P., Rabouille C., Watson R., Hunte F., Watzele G., Berger E. G., Warren G. Kin recognition between medial Golgi enzymes in HeLa cells. EMBO J. 1994 Feb 1;13(3):562–574. doi: 10.1002/j.1460-2075.1994.tb06294.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nilsson T., Rabouille C., Hui N., Watson R., Warren G. The role of the membrane-spanning domain and stalk region of N-acetylglucosaminyltransferase I in retention, kin recognition and structural maintenance of the Golgi apparatus in HeLa cells. J Cell Sci. 1996 Jul;109(Pt 7):1975–1989. doi: 10.1242/jcs.109.7.1975. [DOI] [PubMed] [Google Scholar]
  32. Pensiero M. N., Hay J. The Hantaan virus M-segment glycoproteins G1 and G2 can be expressed independently. J Virol. 1992 Apr;66(4):1907–1914. doi: 10.1128/jvi.66.4.1907-1914.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Persson R., Pettersson R. F. Formation and intracellular transport of a heterodimeric viral spike protein complex. J Cell Biol. 1991 Jan;112(2):257–266. doi: 10.1083/jcb.112.2.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pettersson R. F. Protein localization and virus assembly at intracellular membranes. Curr Top Microbiol Immunol. 1991;170:67–106. doi: 10.1007/978-3-642-76389-2_3. [DOI] [PubMed] [Google Scholar]
  35. Ponnambalam S., Rabouille C., Luzio J. P., Nilsson T., Warren G. The TGN38 glycoprotein contains two non-overlapping signals that mediate localization to the trans-Golgi network. J Cell Biol. 1994 Apr;125(2):253–268. doi: 10.1083/jcb.125.2.253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Rönkä H., Hildén P., Von Bonsdorff C. H., Kuismanen E. Homodimeric association of the spike glycoproteins G1 and G2 of Uukuniemi virus. Virology. 1995 Aug 1;211(1):241–250. doi: 10.1006/viro.1995.1397. [DOI] [PubMed] [Google Scholar]
  39. Rönnholm R. Localization to the Golgi complex of Uukuniemi virus glycoproteins G1 and G2 expressed from cloned cDNAs. J Virol. 1992 Jul;66(7):4525–4531. doi: 10.1128/jvi.66.7.4525-4531.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Rönnholm R., Pettersson R. F. Complete nucleotide sequence of the M RNA segment of Uukuniemi virus encoding the membrane glycoproteins G1 and G2. Virology. 1987 Sep;160(1):191–202. doi: 10.1016/0042-6822(87)90060-2. [DOI] [PubMed] [Google Scholar]
  41. Schmidt M. F. Fatty acylation of proteins. Biochim Biophys Acta. 1989 Dec 6;988(3):411–426. doi: 10.1016/0304-4157(89)90013-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schäfer W., Stroh A., Berghöfer S., Seiler J., Vey M., Kruse M. L., Kern H. F., Klenk H. D., Garten W. Two independent targeting signals in the cytoplasmic domain determine trans-Golgi network localization and endosomal trafficking of the proprotein convertase furin. EMBO J. 1995 Jun 1;14(11):2424–2435. doi: 10.1002/j.1460-2075.1995.tb07240.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Shin J., Dunbrack R. L., Jr, Lee S., Strominger J. L. Signals for retention of transmembrane proteins in the endoplasmic reticulum studied with CD4 truncation mutants. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1918–1922. doi: 10.1073/pnas.88.5.1918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Slusarewicz P., Nilsson T., Hui N., Watson R., Warren G. Isolation of a matrix that binds medial Golgi enzymes. J Cell Biol. 1994 Feb;124(4):405–413. doi: 10.1083/jcb.124.4.405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tang B. L., Low S. H., Wong S. H., Hong W. Cell type differences in Golgi retention signals for transmembrane proteins. Eur J Cell Biol. 1995 Apr;66(4):365–374. [PubMed] [Google Scholar]
  46. Tang B. L., Wong S. H., Low S. H., Hong W. The transmembrane domain of N-glucosaminyltransferase I contains a Golgi retention signal. J Biol Chem. 1992 May 15;267(14):10122–10126. [PubMed] [Google Scholar]
  47. Ulmanen I., Seppälä P., Pettersson R. F. In vitro translation of Uukuniemi virus-specific RNAs: identification of a nonstructural protein and a precursor to the membrane glycoproteins. J Virol. 1981 Jan;37(1):72–79. doi: 10.1128/jvi.37.1.72-79.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Veit M., Kretzschmar E., Kuroda K., Garten W., Schmidt M. F., Klenk H. D., Rott R. Site-specific mutagenesis identifies three cysteine residues in the cytoplasmic tail as acylation sites of influenza virus hemagglutinin. J Virol. 1991 May;65(5):2491–2500. doi: 10.1128/jvi.65.5.2491-2500.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Velasco A., Hendricks L., Moremen K. W., Tulsiani D. R., Touster O., Farquhar M. G. Cell type-dependent variations in the subcellular distribution of alpha-mannosidase I and II. J Cell Biol. 1993 Jul;122(1):39–51. doi: 10.1083/jcb.122.1.39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Weisz O. A., Swift A. M., Machamer C. E. Oligomerization of a membrane protein correlates with its retention in the Golgi complex. J Cell Biol. 1993 Sep;122(6):1185–1196. doi: 10.1083/jcb.122.6.1185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Yang C., Compans R. W. Palmitoylation of the murine leukemia virus envelope glycoprotein transmembrane subunits. Virology. 1996 Jul 1;221(1):87–97. doi: 10.1006/viro.1996.0355. [DOI] [PubMed] [Google Scholar]
  52. Zurcher T., Luo G., Palese P. Mutations at palmitylation sites of the influenza virus hemagglutinin affect virus formation. J Virol. 1994 Sep;68(9):5748–5754. doi: 10.1128/jvi.68.9.5748-5754.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES