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. 1987 Sep;6(9):2683–2691. doi: 10.1002/j.1460-2075.1987.tb02560.x

Deletion analysis of the internal signal-anchor domain of the human asialoglycoprotein receptor H1.

M Spiess 1, C Handschin 1
PMCID: PMC553690  PMID: 3678203

Abstract

The human asialoglycoprotein receptor H1 is a single-spanning membrane protein with the amino terminus facing the cytoplasm and the carboxy terminus exposed on the exoplasmic side of the plasma membrane. It has been shown earlier that the transmembrane segment, residues 38-65, functions as an internal signal directing protein synthesis to the endoplasmic reticulum and initiating membrane insertion. This process is co-translational and mediated by signal recognition particle (SRP). To identify subsegments within this region containing the signal information, we prepared deletion mutants at the level of the cDNA and analysed them in a wheat germ in vitro translation system with microsomes as the target membrane. Insertion and membrane anchoring were judged by the glycosylation of the protein, its resistance to exogenous protease and the extent to which it can be extracted from the microsomes by alkaline treatment. It was found that very small deletions already reduce the stability of membrane anchoring. However, nearly half of the transmembrane domain can be deleted, both from the amino-terminal and from the carboxy-terminal side, without completely abolishing membrane insertion. Several mutants, although not inserted, still interact with SRP. The results support the notion that the main feature of a signal sequence is a hydrophobic stretch of sufficient length (10-12 residues in our sequence), and indicate that recognition by SRP is not sufficient for membrane insertion.

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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. Anderson C. W., Straus J. W., Dudock B. S. Preparation of a cell-free protein-synthesizing system from wheat germ. Methods Enzymol. 1983;101:635–644. doi: 10.1016/0076-6879(83)01044-7. [DOI] [PubMed] [Google Scholar]
  4. Anderson D. J., Mostov K. E., Blobel G. Mechanisms of integration of de novo-synthesized polypeptides into membranes: signal-recognition particle is required for integration into microsomal membranes of calcium ATPase and of lens MP26 but not of cytochrome b5. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7249–7253. doi: 10.1073/pnas.80.23.7249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ashwell G., Harford J. Carbohydrate-specific receptors of the liver. Annu Rev Biochem. 1982;51:531–554. doi: 10.1146/annurev.bi.51.070182.002531. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. 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]
  9. Davis N. G., Boeke J. D., Model P. Fine structure of a membrane anchor domain. J Mol Biol. 1985 Jan 5;181(1):111–121. doi: 10.1016/0022-2836(85)90329-8. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Garoff H. Using recombinant DNA techniques to study protein targeting in the eucaryotic cell. Annu Rev Cell Biol. 1985;1:403–445. doi: 10.1146/annurev.cb.01.110185.002155. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Kaiser C. A., Preuss D., Grisafi P., Botstein D. Many random sequences functionally replace the secretion signal sequence of yeast invertase. Science. 1987 Jan 16;235(4786):312–317. doi: 10.1126/science.3541205. [DOI] [PubMed] [Google Scholar]
  15. Krieg P. A., Melton D. A. Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res. 1984 Sep 25;12(18):7057–7070. doi: 10.1093/nar/12.18.7057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. 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]
  20. Mize N. K., Andrews D. W., Lingappa V. R. A stop transfer sequence recognizes receptors for nascent chain translocation across the endoplasmic reticulum membrane. Cell. 1986 Dec 5;47(5):711–719. doi: 10.1016/0092-8674(86)90514-3. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Schwartz A. L. The hepatic asialoglycoprotein receptor. CRC Crit Rev Biochem. 1984;16(3):207–233. doi: 10.3109/10409238409108716. [DOI] [PubMed] [Google Scholar]
  23. Sivasubramanian N., Nayak D. P. Mutational analysis of the signal-anchor domain of influenza virus neuraminidase. Proc Natl Acad Sci U S A. 1987 Jan;84(1):1–5. doi: 10.1073/pnas.84.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Spiess M., Lodish H. F. Sequence of a second human asialoglycoprotein receptor: conservation of two receptor genes during evolution. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6465–6469. doi: 10.1073/pnas.82.19.6465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Spiess M., Schwartz A. L., Lodish H. F. Sequence of human asialoglycoprotein receptor cDNA. An internal signal sequence for membrane insertion. J Biol Chem. 1985 Feb 25;260(4):1979–1982. [PubMed] [Google Scholar]
  27. Walter P., Blobel G. Preparation of microsomal membranes for cotranslational protein translocation. Methods Enzymol. 1983;96:84–93. doi: 10.1016/s0076-6879(83)96010-x. [DOI] [PubMed] [Google Scholar]
  28. Walter P., Blobel G. Signal recognition particle: a ribonucleoprotein required for cotranslational translocation of proteins, isolation and properties. Methods Enzymol. 1983;96:682–691. doi: 10.1016/s0076-6879(83)96057-3. [DOI] [PubMed] [Google Scholar]
  29. Walter P., Blobel G. Translocation of proteins across the endoplasmic reticulum III. Signal recognition protein (SRP) causes signal sequence-dependent and site-specific arrest of chain elongation that is released by microsomal membranes. J Cell Biol. 1981 Nov;91(2 Pt 1):557–561. doi: 10.1083/jcb.91.2.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Walter P., Gilmore R., Blobel G. Protein translocation across the endoplasmic reticulum. Cell. 1984 Aug;38(1):5–8. doi: 10.1016/0092-8674(84)90520-8. [DOI] [PubMed] [Google Scholar]
  31. Watson M. E. Compilation of published signal sequences. Nucleic Acids Res. 1984 Jul 11;12(13):5145–5164. doi: 10.1093/nar/12.13.5145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. 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]

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