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. 1989 Nov 1;109(5):2023–2032. doi: 10.1083/jcb.109.5.2023

Transposition of domains between the M2 and HN viral membrane proteins results in polypeptides which can adopt more than one membrane orientation

PMCID: PMC2115837  PMID: 2553741

Abstract

The influenza A virus M2 polypeptide is a small integral membrane protein that does not contain a cleaved signal sequence, but is unusual in that it assumes the membrane orientation of a class I integral membrane protein with an NH2-terminal ectodomain and a COOH-terminal cytoplasmic tail. To determine the domains of M2 involved in specifying membrane orientation, hybrid genes were constructed and expressed in which regions of the M2 protein were linked to portions of the paramyxovirus HN and SH proteins, two class II integral membrane proteins that adopt the opposite orientation in membranes from M2. A hybrid protein (MgMH) consisting of the M2 NH2-terminal and membrane- spanning domains linked precisely to the HN COOH-terminal ectodomain was found in cells in two forms: integrated into membranes in the M2 topology or completely translocated across the endoplasmic reticulum membrane and ultimately secreted from the cell. The finding of a soluble form suggested that in this hybrid protein the anchor function of the M2 signal/anchor domain can be overridden. A second hybrid which contained the M2 NH2 terminus linked to the HN signal anchor and ectodomain (MgHH) was found in both the M2 and the HN orientation, suggesting that the M2 NH2 terminus was capable of reversing the topology of a class II membrane protein. The exchange of the M2 signal/anchor domain with that of SH resulted in a hybrid protein which assumed only the M2 topology. Thus, all these data suggest that the NH2- terminal 24 residues to M2 are important for directing the unusual membrane topology of the M2 protein. These data are discussed in relationship to the loop model for insertion of proteins into membranes and the role of charged residues as a factor in determining orientation.

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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. Anderson D. J., Blobel G. Immunoprecipitation of proteins from cell-free translations. Methods Enzymol. 1983;96:111–120. doi: 10.1016/s0076-6879(83)96012-3. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Brown D. J., Hogue B. G., Nayak D. P. Redundancy of signal and anchor functions in the NH2-terminal uncharged region of influenza virus neuraminidase, a class II membrane glycoprotein. J Virol. 1988 Oct;62(10):3824–3831. doi: 10.1128/jvi.62.10.3824-3831.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Haeuptle M. T., Flint N., Gough N. M., Dobberstein B. A tripartite structure of the signals that determine protein insertion into the endoplasmic reticulum membrane. J Cell Biol. 1989 Apr;108(4):1227–1236. doi: 10.1083/jcb.108.4.1227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hiebert S. W., Paterson R. G., Lamb R. A. Identification and predicted sequence of a previously unrecognized small hydrophobic protein, SH, of the paramyxovirus simian virus 5. J Virol. 1985 Sep;55(3):744–751. doi: 10.1128/jvi.55.3.744-751.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hiebert S. W., Richardson C. D., Lamb R. A. Cell surface expression and orientation in membranes of the 44-amino-acid SH protein of simian virus 5. J Virol. 1988 Jul;62(7):2347–2357. doi: 10.1128/jvi.62.7.2347-2357.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hull J. D., Gilmore R., Lamb R. A. Integration of a small integral membrane protein, M2, of influenza virus into the endoplasmic reticulum: analysis of the internal signal-anchor domain of a protein with an ectoplasmic NH2 terminus. J Cell Biol. 1988 May;106(5):1489–1498. doi: 10.1083/jcb.106.5.1489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lamb R. A., Choppin P. W. Synthesis of influenza virus proteins in infected cells: translation of viral polypeptides, including three P polypeptides, from RNA produced by primary transcription. Virology. 1976 Oct 15;74(2):504–519. doi: 10.1016/0042-6822(76)90356-1. [DOI] [PubMed] [Google Scholar]
  13. Lamb R. A., Choppin P. W. The synthesis of Sendai virus polypeptides in infected cells. III. Phosphorylation of polypeptides. Virology. 1977 Sep;81(2):382–397. doi: 10.1016/0042-6822(77)90154-4. [DOI] [PubMed] [Google Scholar]
  14. Lamb R. A., Etkind P. R., Choppin P. W. Evidence for a ninth influenza viral polypeptide. Virology. 1978 Nov;91(1):60–78. doi: 10.1016/0042-6822(78)90355-0. [DOI] [PubMed] [Google Scholar]
  15. Lamb R. A., Lai C. J. Spliced and unspliced messenger RNAs synthesized from cloned influenza virus M DNA in an SV40 vector: expression of the influenza virus membrane protein (M1). Virology. 1982 Dec;123(2):237–256. doi: 10.1016/0042-6822(82)90258-6. [DOI] [PubMed] [Google Scholar]
  16. Lamb R. A., Zebedee S. L., Richardson C. D. Influenza virus M2 protein is an integral membrane protein expressed on the infected-cell surface. Cell. 1985 Mar;40(3):627–633. doi: 10.1016/0092-8674(85)90211-9. [DOI] [PubMed] [Google Scholar]
  17. Lipp J., Dobberstein B. Signal and membrane anchor functions overlap in the type II membrane protein I gamma CAT. J Cell Biol. 1988 Jun;106(6):1813–1820. doi: 10.1083/jcb.106.6.1813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lipp J., Dobberstein B. The membrane-spanning segment of invariant chain (I gamma) contains a potentially cleavable signal sequence. Cell. 1986 Sep 26;46(7):1103–1112. doi: 10.1016/0092-8674(86)90710-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Monier S., Van Luc P., Kreibich G., Sabatini D. D., Adesnik M. Signals for the incorporation and orientation of cytochrome P450 in the endoplasmic reticulum membrane. J Cell Biol. 1988 Aug;107(2):457–470. doi: 10.1083/jcb.107.2.457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Parks G. D., Baker J. C., Palmenberg A. C. Proteolytic cleavage of encephalomyocarditis virus capsid region substrates by precursors to the 3C enzyme. J Virol. 1989 Mar;63(3):1054–1058. doi: 10.1128/jvi.63.3.1054-1058.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Paterson R. G., Hiebert S. W., Lamb R. A. Expression at the cell surface of biologically active fusion and hemagglutinin/neuraminidase proteins of the paramyxovirus simian virus 5 from cloned cDNA. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7520–7524. doi: 10.1073/pnas.82.22.7520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Paterson R. G., Lamb R. A. Ability of the hydrophobic fusion-related external domain of a paramyxovirus F protein to act as a membrane anchor. Cell. 1987 Feb 13;48(3):441–452. doi: 10.1016/0092-8674(87)90195-4. [DOI] [PubMed] [Google Scholar]
  23. Pipas J. M., Peden K. W., Nathans D. Mutational analysis of simian virus 40 T antigen: isolation and characterization of mutants with deletions in the T-antigen gene. Mol Cell Biol. 1983 Feb;3(2):203–213. doi: 10.1128/mcb.3.2.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rose J. K., Welch W. J., Sefton B. M., Esch F. S., Ling N. C. Vesicular stomatitis virus glycoprotein is anchored in the viral membrane by a hydrophobic domain near the COOH terminus. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3884–3888. doi: 10.1073/pnas.77.7.3884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Russell D. W., Schneider W. J., Yamamoto T., Luskey K. L., Brown M. S., Goldstein J. L. Domain map of the LDL receptor: sequence homology with the epidermal growth factor precursor. Cell. 1984 Jun;37(2):577–585. doi: 10.1016/0092-8674(84)90388-x. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Sakaguchi M., Mihara K., Sato R. Signal recognition particle is required for co-translational insertion of cytochrome P-450 into microsomal membranes. Proc Natl Acad Sci U S A. 1984 Jun;81(11):3361–3364. doi: 10.1073/pnas.81.11.3361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schatzman R. C., Evan G. I., Privalsky M. L., Bishop J. M. Orientation of the v-erb-B gene product in the plasma membrane. Mol Cell Biol. 1986 Apr;6(4):1329–1333. doi: 10.1128/mcb.6.4.1329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schneider C., Owen M. J., Banville D., Williams J. G. Primary structure of human transferrin receptor deduced from the mRNA sequence. Nature. 1984 Oct 18;311(5987):675–678. doi: 10.1038/311675b0. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Simon K., Lingappa V. R., Ganem D. Secreted hepatitis B surface antigen polypeptides are derived from a transmembrane precursor. J Cell Biol. 1988 Dec;107(6 Pt 1):2163–2168. doi: 10.1083/jcb.107.6.2163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Spiess M., Handschin C. Deletion analysis of the internal signal-anchor domain of the human asialoglycoprotein receptor H1. EMBO J. 1987 Sep;6(9):2683–2691. doi: 10.1002/j.1460-2075.1987.tb02560.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Walter P., Lingappa V. R. Mechanism of protein translocation across the endoplasmic reticulum membrane. Annu Rev Cell Biol. 1986;2:499–516. doi: 10.1146/annurev.cb.02.110186.002435. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Williams M. A., Lamb R. A. Determination of the orientation of an integral membrane protein and sites of glycosylation by oligonucleotide-directed mutagenesis: influenza B virus NB glycoprotein lacks a cleavable signal sequence and has an extracellular NH2-terminal region. Mol Cell Biol. 1986 Dec;6(12):4317–4328. doi: 10.1128/mcb.6.12.4317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zebedee S. L., Lamb R. A. Influenza A virus M2 protein: monoclonal antibody restriction of virus growth and detection of M2 in virions. J Virol. 1988 Aug;62(8):2762–2772. doi: 10.1128/jvi.62.8.2762-2772.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zebedee S. L., Richardson C. D., Lamb R. A. Characterization of the influenza virus M2 integral membrane protein and expression at the infected-cell surface from cloned cDNA. J Virol. 1985 Nov;56(2):502–511. doi: 10.1128/jvi.56.2.502-511.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zerial M., Huylebroeck D., Garoff H. Foreign transmembrane peptides replacing the internal signal sequence of transferrin receptor allow its translocation and membrane binding. Cell. 1987 Jan 16;48(1):147–155. doi: 10.1016/0092-8674(87)90365-5. [DOI] [PubMed] [Google Scholar]
  41. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]

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