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. 1990 Sep 1;111(3):867–876. doi: 10.1083/jcb.111.3.867

The signal sequence of the p62 protein of Semliki Forest virus is involved in initiation but not in completing chain translocation

PMCID: PMC2116283  PMID: 2391367

Abstract

So far it has been demonstrated that the signal sequence of proteins which are made at the ER functions both at the level of protein targeting to the ER and in initiation of chain translocation across the ER membrane. However, its possible role in completing the process of chain transfer (see Singer, S. J., P. A. Maher, and M. P. Yaffe. Proc. Natl. Acad. Sci. USA. 1987. 84:1015-1019) has remained elusive. In this work we show that the p62 protein of Semliki Forest virus contains an uncleaved signal sequence at its NH2-terminus and that this becomes glycosylated early during synthesis and translocation of the p62 polypeptide. As the glycosylation of the signal sequence most likely occurs after its release from the ER membrane our results suggest that this region has no role in completing the transfer process.

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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. Batenburg A. M., Brasseur R., Ruysschaert J. M., van Scharrenburg G. J., Slotboom A. J., Demel R. A., de Kruijff B. Characterization of the interfacial behavior and structure of the signal sequence of Escherichia coli outer membrane pore protein PhoE. J Biol Chem. 1988 Mar 25;263(9):4202–4207. [PubMed] [Google Scholar]
  3. Bergman L. W., Kuehl W. M. Addition of glucosamine and mannose to nascent immunoglobulin heavy chains. Biochemistry. 1977 Oct 4;16(20):4490–4497. doi: 10.1021/bi00639a025. [DOI] [PubMed] [Google Scholar]
  4. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [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., Sabatini D. D. Controlled proteolysis of nascent polypeptides in rat liver cell fractions. I. Location of the polypeptides within ribosomes. J Cell Biol. 1970 Apr;45(1):130–145. doi: 10.1083/jcb.45.1.130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bonatti S., Migliaccio G., Blobel G., Walter P. Role of signal recognition particle in the membrane assembly of Sindbis viral glycoproteins. Eur J Biochem. 1984 May 2;140(3):499–502. doi: 10.1111/j.1432-1033.1984.tb08130.x. [DOI] [PubMed] [Google Scholar]
  8. Borgese N., Mok W., Kreibich G., Sabatini D. D. Ribosomal-membrane interaction: in vitro binding of ribosomes to microsomal membranes. J Mol Biol. 1974 Sep 25;88(3):559–580. doi: 10.1016/0022-2836(74)90408-2. [DOI] [PubMed] [Google Scholar]
  9. Bos T. J., Davis A. R., Nayak D. P. NH2-terminal hydrophobic region of influenza virus neuraminidase provides the signal function in translocation. Proc Natl Acad Sci U S A. 1984 Apr;81(8):2327–2331. doi: 10.1073/pnas.81.8.2327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Briggs M. S., Cornell D. G., Dluhy R. A., Gierasch L. M. Conformations of signal peptides induced by lipids suggest initial steps in protein export. Science. 1986 Jul 11;233(4760):206–208. doi: 10.1126/science.2941862. [DOI] [PubMed] [Google Scholar]
  11. Briggs M. S., Gierasch L. M., Zlotnick A., Lear J. D., DeGrado W. F. In vivo function and membrane binding properties are correlated for Escherichia coli lamB signal peptides. Science. 1985 May 31;228(4703):1096–1099. doi: 10.1126/science.3158076. [DOI] [PubMed] [Google Scholar]
  12. Chang A. C., Nunberg J. H., Kaufman R. J., Erlich H. A., Schimke R. T., Cohen S. N. Phenotypic expression in E. coli of a DNA sequence coding for mouse dihydrofolate reductase. Nature. 1978 Oct 19;275(5681):617–624. doi: 10.1038/275617a0. [DOI] [PubMed] [Google Scholar]
  13. Chang G. J., Trent D. W. Nucleotide sequence of the genome region encoding the 26S mRNA of eastern equine encephalomyelitis virus and the deduced amino acid sequence of the viral structural proteins. J Gen Virol. 1987 Aug;68(Pt 8):2129–2142. doi: 10.1099/0022-1317-68-8-2129. [DOI] [PubMed] [Google Scholar]
  14. Cornell D. G., Dluhy R. A., Briggs M. S., McKnight C. J., Gierasch L. M. Conformations and orientations of a signal peptide interacting with phospholipid monolayers. Biochemistry. 1989 Apr 4;28(7):2789–2797. doi: 10.1021/bi00433a008. [DOI] [PubMed] [Google Scholar]
  15. Crouse G. F., Simonsen C. C., McEwan R. N., Schimke R. T. Structure of amplified normal and variant dihydrofolate reductase genes in mouse sarcoma S180 cells. J Biol Chem. 1982 Jul 10;257(13):7887–7897. [PubMed] [Google Scholar]
  16. 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]
  17. Dalgarno L., Rice C. M., Strauss J. H. Ross River virus 26 s RNA: complete nucleotide sequence and deduced sequence of the encoded structural proteins. Virology. 1983 Aug;129(1):170–187. doi: 10.1016/0042-6822(83)90404-x. [DOI] [PubMed] [Google Scholar]
  18. Emr S. D., Silhavy T. J. Importance of secondary structure in the signal sequence for protein secretion. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4599–4603. doi: 10.1073/pnas.80.15.4599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Engelman D. M., Steitz T. A. The spontaneous insertion of proteins into and across membranes: the helical hairpin hypothesis. Cell. 1981 Feb;23(2):411–422. doi: 10.1016/0092-8674(81)90136-7. [DOI] [PubMed] [Google Scholar]
  20. Garoff H., Frischauf A. M., Simons K., Lehrach H., Delius H. Nucleotide sequence of cdna coding for Semliki Forest virus membrane glycoproteins. Nature. 1980 Nov 20;288(5788):236–241. doi: 10.1038/288236a0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. 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]
  24. Geetha-Habib M., Noiva R., Kaplan H. A., Lennarz W. J. Glycosylation site binding protein, a component of oligosaccharyl transferase, is highly similar to three other 57 kd luminal proteins of the ER. Cell. 1988 Sep 23;54(7):1053–1060. doi: 10.1016/0092-8674(88)90120-1. [DOI] [PubMed] [Google Scholar]
  25. Gielkens A. L., Van Zaane D., Bloemers H. P., Bloemendal H. Synthesis of Rauscher murine leukemia virus-specific polypeptides in vitro. Proc Natl Acad Sci U S A. 1976 Feb;73(2):356–360. doi: 10.1073/pnas.73.2.356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Gilmore R., Blobel G. Translocation of secretory proteins across the microsomal membrane occurs through an environment accessible to aqueous perturbants. Cell. 1985 Sep;42(2):497–505. doi: 10.1016/0092-8674(85)90107-2. [DOI] [PubMed] [Google Scholar]
  27. Glabe C. G., Hanover J. A., Lennarz W. J. Glycosylation of ovalbumin nascent chains. The spatial relationship between translation and glycosylation. J Biol Chem. 1980 Oct 10;255(19):9236–9242. [PubMed] [Google Scholar]
  28. Hahn C. S., Strauss E. G., Strauss J. H. Sequence analysis of three Sindbis virus mutants temperature-sensitive in the capsid protein autoprotease. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4648–4652. doi: 10.1073/pnas.82.14.4648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hashimoto K., Erdei S., Keränen S., Saraste J., Käriäinen L. Evidence for a separate signal sequence for the carboxy-terminal envelope glycoprotein E1 of Semliki forest virus. J Virol. 1981 Apr;38(1):34–40. doi: 10.1128/jvi.38.1.34-40.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Jackson R. J., Hunt T. Preparation and use of nuclease-treated rabbit reticulocyte lysates for the translation of eukaryotic messenger RNA. Methods Enzymol. 1983;96:50–74. doi: 10.1016/s0076-6879(83)96008-1. [DOI] [PubMed] [Google Scholar]
  31. Kaderbhai M. A., Austen B. M. Dog pancreatic microsomal-membrane polypeptides analysed by two-dimensional gel electrophoresis. Biochem J. 1984 Jan 1;217(1):145–157. doi: 10.1042/bj2170145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kahn M., Kolter R., Thomas C., Figurski D., Meyer R., Remaut E., Helinski D. R. Plasmid cloning vehicles derived from plasmids ColE1, F, R6K, and RK2. Methods Enzymol. 1979;68:268–280. doi: 10.1016/0076-6879(79)68019-9. [DOI] [PubMed] [Google Scholar]
  33. Kendall D. A., Bock S. C., Kaiser E. T. Idealization of the hydrophobic segment of the alkaline phosphatase signal peptide. Nature. 1986 Jun 12;321(6071):706–708. doi: 10.1038/321706a0. [DOI] [PubMed] [Google Scholar]
  34. Kinney R. M., Johnson B. J., Brown V. L., Trent D. W. Nucleotide sequence of the 26 S mRNA of the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus and deduced sequence of the encoded structural proteins. Virology. 1986 Jul 30;152(2):400–413. doi: 10.1016/0042-6822(86)90142-x. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Liebhaber S. A., Begley K. A. Structural and evolutionary analysis of the two chimpanzee alpha-globin mRNAs. Nucleic Acids Res. 1983 Dec 20;11(24):8915–8929. doi: 10.1093/nar/11.24.8915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Lipp J., Dobberstein B. Signal recognition particle-dependent membrane insertion of mouse invariant chain: a membrane-spanning protein with a cytoplasmically exposed amino terminus. J Cell Biol. 1986 Jun;102(6):2169–2175. doi: 10.1083/jcb.102.6.2169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Lodish H. F. Transport of secretory and membrane glycoproteins from the rough endoplasmic reticulum to the Golgi. A rate-limiting step in protein maturation and secretion. J Biol Chem. 1988 Feb 15;263(5):2107–2110. [PubMed] [Google Scholar]
  40. Malkin L. I., Rich A. Partial resistance of nascent polypeptide chains to proteolytic digestion due to ribosomal shielding. J Mol Biol. 1967 Jun 14;26(2):329–346. doi: 10.1016/0022-2836(67)90301-4. [DOI] [PubMed] [Google Scholar]
  41. Mayer T., Tamura T., Falk M., Niemann H. Membrane integration and intracellular transport of the coronavirus glycoprotein E1, a class III membrane glycoprotein. J Biol Chem. 1988 Oct 15;263(29):14956–14963. doi: 10.1016/S0021-9258(18)68131-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Mayne J. T., Rice C. M., Strauss E. G., Hunkapiller M. W., Strauss J. H. Biochemical studies of the maturation of the small Sindbis virus glycoprotein E3. Virology. 1984 Apr 30;134(2):338–357. doi: 10.1016/0042-6822(84)90302-7. [DOI] [PubMed] [Google Scholar]
  43. Melancon P., Garoff H. Processing of the Semliki Forest virus structural polyprotein: role of the capsid protease. J Virol. 1987 May;61(5):1301–1309. doi: 10.1128/jvi.61.5.1301-1309.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Melancon P., Garoff H. Reinitiation of translocation in the Semliki Forest virus structural polyprotein: identification of the signal for the E1 glycoprotein. EMBO J. 1986 Jul;5(7):1551–1560. doi: 10.1002/j.1460-2075.1986.tb04396.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Nesmeyanova M. A. On the possible participation of acid phospholipids in the translocation of secreted proteins through the bacterial cytoplasmic membrane. FEBS Lett. 1982 Jun 7;142(2):189–193. doi: 10.1016/0014-5793(82)80131-2. [DOI] [PubMed] [Google Scholar]
  46. Nunberg J. H., Kaufman R. J., Chang A. C., Cohen S. N., Schimke R. T. Structure and genomic organization of the mouse dihydrofolate reductase gene. Cell. 1980 Feb;19(2):355–364. doi: 10.1016/0092-8674(80)90510-3. [DOI] [PubMed] [Google Scholar]
  47. Randall L. L. Translocation of domains of nascent periplasmic proteins across the cytoplasmic membrane is independent of elongation. Cell. 1983 May;33(1):231–240. doi: 10.1016/0092-8674(83)90352-5. [DOI] [PubMed] [Google Scholar]
  48. Rapoport T. A. Protein translocation across and integration into membranes. CRC Crit Rev Biochem. 1986;20(1):73–137. doi: 10.3109/10409238609115901. [DOI] [PubMed] [Google Scholar]
  49. Remaut E., Tsao H., Fiers W. Improved plasmid vectors with a thermoinducible expression and temperature-regulated runaway replication. Gene. 1983 Apr;22(1):103–113. doi: 10.1016/0378-1119(83)90069-0. [DOI] [PubMed] [Google Scholar]
  50. Rice C. M., Strauss J. H. Nucleotide sequence of the 26S mRNA of Sindbis virus and deduced sequence of the encoded virus structural proteins. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2062–2066. doi: 10.1073/pnas.78.4.2062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Robinson A., Kaderbhai M. A., Austen B. M. Identification of signal sequence binding proteins integrated into the rough endoplasmic reticulum membrane. Biochem J. 1987 Mar 15;242(3):767–777. doi: 10.1042/bj2420767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. Rothman J. E. Polypeptide chain binding proteins: catalysts of protein folding and related processes in cells. Cell. 1989 Nov 17;59(4):591–601. doi: 10.1016/0092-8674(89)90005-6. [DOI] [PubMed] [Google Scholar]
  54. Shaw A. S., Rottier P. J., Rose J. K. Evidence for the loop model of signal-sequence insertion into the endoplasmic reticulum. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7592–7596. doi: 10.1073/pnas.85.20.7592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Singer S. J., Maher P. A., Yaffe M. P. On the transfer of integral proteins into membranes. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1960–1964. doi: 10.1073/pnas.84.7.1960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Singer S. J., Maher P. A., Yaffe M. P. On the translocation of proteins across membranes. Proc Natl Acad Sci U S A. 1987 Feb;84(4):1015–1019. doi: 10.1073/pnas.84.4.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Smith W. P., Tai P. C., Davis B. D. Nascent peptide as sole attachment of polysomes to membranes in bacteria. Proc Natl Acad Sci U S A. 1978 Feb;75(2):814–817. doi: 10.1073/pnas.75.2.814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Spiess M., Lodish H. F. An internal signal sequence: the asialoglycoprotein receptor membrane anchor. Cell. 1986 Jan 17;44(1):177–185. doi: 10.1016/0092-8674(86)90496-4. [DOI] [PubMed] [Google Scholar]
  59. Sutcliffe J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):77–90. doi: 10.1101/sqb.1979.043.01.013. [DOI] [PubMed] [Google Scholar]
  60. Tillmann U., Günther R., Schweden J., Bause E. Subcellular location of enzymes involved in the N-glycosylation and processing of asparagine-linked oligosaccharides in Saccharomyces cerevisiae. Eur J Biochem. 1987 Feb 2;162(3):635–642. doi: 10.1111/j.1432-1033.1987.tb10685.x. [DOI] [PubMed] [Google Scholar]
  61. Wessels H. P., Spiess M. Insertion of a multispanning membrane protein occurs sequentially and requires only one signal sequence. Cell. 1988 Oct 7;55(1):61–70. doi: 10.1016/0092-8674(88)90009-8. [DOI] [PubMed] [Google Scholar]
  62. Wickner W. T., Lodish H. F. Multiple mechanisms of protein insertion into and across membranes. Science. 1985 Oct 25;230(4724):400–407. doi: 10.1126/science.4048938. [DOI] [PubMed] [Google Scholar]
  63. Wiedmann M., Kurzchalia T. V., Hartmann E., Rapoport T. A. A signal sequence receptor in the endoplasmic reticulum membrane. 1987 Aug 27-Sep 2Nature. 328(6133):830–833. doi: 10.1038/328830a0. [DOI] [PubMed] [Google Scholar]
  64. Wier M., Edidin M. Constraint of the translational diffusion of a membrane glycoprotein by its external domains. Science. 1988 Oct 21;242(4877):412–414. doi: 10.1126/science.3175663. [DOI] [PubMed] [Google Scholar]
  65. Zerial M., Melancon P., Schneider C., Garoff H. The transmembrane segment of the human transferrin receptor functions as a signal peptide. EMBO J. 1986 Jul;5(7):1543–1550. doi: 10.1002/j.1460-2075.1986.tb04395.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. de Curtis I., Simons K. Dissection of Semliki Forest virus glycoprotein delivery from the trans-Golgi network to the cell surface in permeabilized BHK cells. Proc Natl Acad Sci U S A. 1988 Nov;85(21):8052–8056. doi: 10.1073/pnas.85.21.8052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. von Heijne G., Blomberg C. Trans-membrane translocation of proteins. The direct transfer model. Eur J Biochem. 1979 Jun;97(1):175–181. doi: 10.1111/j.1432-1033.1979.tb13100.x. [DOI] [PubMed] [Google Scholar]

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