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. 1988 Jun 1;106(6):1813–1820. doi: 10.1083/jcb.106.6.1813

Signal and membrane anchor functions overlap in the type II membrane protein I gamma CAT

PMCID: PMC2115136  PMID: 3290220

Abstract

I gamma CAT is a hybrid protein that inserts into the membrane of the endoplasmic reticulum as a type II membrane protein. These proteins span the membrane once and expose the NH2-terminal end on the cytoplasmic side and the COOH terminus on the exoplasmic side. I gamma CAT has a single hydrophobic segment of 30 amino acid residues that functions as a signal for membrane insertion and anchoring. The signal- anchor region in I gamma CAT was analyzed by deletion mutagenesis from its COOH-terminal end (delta C mutants). The results show that the 13 amino acid residues on the amino-terminal side of the hydrophobic segment are not sufficient for membrane insertion and translocation. Mutant proteins with at least 16 of the hydrophobic residues are inserted into the membrane, glycosylated, and partially proteolytically processed by a microsomal protease (signal peptidase). The degree of processing varies between different delta C mutants. Mutant proteins retaining 20 or more of the hydrophobic amino acid residues can span the membrane like the parent I gamma CAT protein and are not proteolytically processed. Our data suggest that in the type II membrane protein I gamma CAT, the signals for membrane insertion and anchoring are overlapping and that hydrophilic amino acid residues at the COOH-terminal end of the hydrophobic segment can influence cleavage by signal peptidase. From this and previous work, we conclude that the function of the signal-anchor sequence in I gamma CAT is determined by three segments: a positively charged NH2 terminus, a hydrophobic core of at least 16 amino acid residues, and the COOH-terminal flanking hydrophilic segment.

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Selected References

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  1. Abrahmsén L., Moks T., Nilsson B., Hellman U., Uhlén M. Analysis of signals for secretion in the staphylococcal protein A gene. EMBO J. 1985 Dec 30;4(13B):3901–3906. doi: 10.1002/j.1460-2075.1985.tb04164.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. 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]
  5. 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]
  6. Chiacchia K. B., Drickamer K. Direct evidence for the transmembrane orientation of the hepatic glycoprotein receptors. J Biol Chem. 1984 Dec 25;259(24):15440–15446. [PubMed] [Google Scholar]
  7. Claesson L., Larhammar D., Rask L., Peterson P. A. cDNA clone for the human invariant gamma chain of class II histocompatibility antigens and its implications for the protein structure. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7395–7399. doi: 10.1073/pnas.80.24.7395. [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., 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]
  10. 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]
  11. Haeuptle M. T., Frank R., Dobberstein B. Translation arrest by oligodeoxynucleotides complementary to mRNA coding sequences yields polypeptides of predetermined length. Nucleic Acids Res. 1986 Feb 11;14(3):1427–1448. doi: 10.1093/nar/14.3.1427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Holland E. C., Leung J. O., Drickamer K. Rat liver asialoglycoprotein receptor lacks a cleavable NH2-terminal signal sequence. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7338–7342. doi: 10.1073/pnas.81.23.7338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jackson R. C., Blobel G. Post-translational cleavage of presecretory proteins with an extract of rough microsomes from dog pancreas containing signal peptidase activity. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5598–5602. doi: 10.1073/pnas.74.12.5598. [DOI] [PMC free article] [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. 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]
  16. Lau J. T., Welply J. K., Shenbagamurthi P., Naider F., Lennarz W. J. Substrate recognition by oligosaccharyl transferase. Inhibition of co-translational glycosylation by acceptor peptides. J Biol Chem. 1983 Dec 25;258(24):15255–15260. [PubMed] [Google Scholar]
  17. Legerski R. J., Hodnett J. L., Gray H. B., Jr Extracellular nucleases of pseudomonas BAL 31. III. Use of the double-strand deoxyriboexonuclease activity as the basis of a convenient method for the mapping of fragments of DNA produced by cleavage with restriction enzymes. Nucleic Acids Res. 1978 May;5(5):1445–1464. doi: 10.1093/nar/5.5.1445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lehnhardt S., Pollitt S., Inouye M. The differential effect on two hybrid proteins of deletion mutations within the hydrophobic region of the Escherichia coli OmpA signal peptide. J Biol Chem. 1987 Feb 5;262(4):1716–1719. [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Markoff L., Lin B. C., Sveda M. M., Lai C. J. Glycosylation and surface expression of the influenza virus neuraminidase requires the N-terminal hydrophobic region. Mol Cell Biol. 1984 Jan;4(1):8–16. doi: 10.1128/mcb.4.1.8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Moreno F., Fowler A. V., Hall M., Silhavy T. J., Zabin I., Schwartz M. A signal sequence is not sufficient to lead beta-galactosidase out of the cytoplasm. Nature. 1980 Jul 24;286(5771):356–359. doi: 10.1038/286356a0. [DOI] [PubMed] [Google Scholar]
  23. Norrander J., Kempe T., Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983 Dec;26(1):101–106. doi: 10.1016/0378-1119(83)90040-9. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Semenza G. Anchoring and biosynthesis of stalked brush border membrane proteins: glycosidases and peptidases of enterocytes and renal tubuli. Annu Rev Cell Biol. 1986;2:255–313. doi: 10.1146/annurev.cb.02.110186.001351. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Strubin M., Mach B., Long E. O. The complete sequence of the mRNA for the HLA-DR-associated invariant chain reveals a polypeptide with an unusual transmembrane polarity. EMBO J. 1984 Apr;3(4):869–872. doi: 10.1002/j.1460-2075.1984.tb01898.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Stueber D., Ibrahimi I., Cutler D., Dobberstein B., Bujard H. A novel in vitro transcription-translation system: accurate and efficient synthesis of single proteins from cloned DNA sequences. EMBO J. 1984 Dec 20;3(13):3143–3148. doi: 10.1002/j.1460-2075.1984.tb02271.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Weinstein J. N., Blumenthal R., van Renswoude J., Kempf C., Klausner R. D. Charge clusters and the orientation of membrane proteins. J Membr Biol. 1982;66(3):203–212. doi: 10.1007/BF01868495. [DOI] [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. von Heijne G. Towards a comparative anatomy of N-terminal topogenic protein sequences. J Mol Biol. 1986 May 5;189(1):239–242. doi: 10.1016/0022-2836(86)90394-3. [DOI] [PubMed] [Google Scholar]

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