Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Feb 15;322(Pt 1):335–342. doi: 10.1042/bj3220335

Secretion of a type II integral membrane protein induced by mutation of the transmembrane segment.

I Lemire 1, C Lazure 1, P Crine 1, G Boileau 1
PMCID: PMC1218196  PMID: 9078281

Abstract

Signal peptide/membrane anchor (SA) domains of type II membrane proteins initiate the translocation of downstream polypeptides across the endoplasmic reticulum (ER) membrane. In contrast with signal peptides, however, SA domains are not cleaved by signal peptidase and thus anchor the protein in the membrane. In the present study we have introduced mutations in the SA domain of neprilysin (neutral endopeptidase-24.11; NEP) to identify structural elements that would favour the processing of SA domains by signal peptidase. Mutants of full-length and truncated (without cytoplasmic domain) protein were constructed by substitution of the sequences SQNS, QQTT or YPGY for VTMI starting at position 15 of the NEP SA domain. In addition, a Pro residue was substituted for Thr at position 16 of the SA domain. The rationale for the use of these sequences was decided from our previous observation that substitution in the NEP SA domain of the sequence SQNS, which is polar and has alpha-helix-breaking potential, could promote SA domain processing under certain conditions (Roy, Chatellard, Lemay, Crine and Boileau (1993) J. Biol. Chem. 268. 2699-2704; Yang. Chatellard, Lazure, Crine and Boileau (1994) Arch. Biochem. Biophys. 315, 382-386). The QQTT sequence is polar but, according to secondary structure predictions, is compatible with the alpha-helix structure of the NEP SA domain. The YPGY sequence and single Pro residue are less polar and have alpha-helix-breaking potential. The predicted effects of these mutations on the structure of the NEP SA domain were confirmed by CD analysis of 42-residue peptides encompassing the hydrophobic segment and flanking regions. Wild-type and mutated proteins were expressed in COS-I cells and their fate (membrane-bound or secreted) was determined by immunoblotting and by endoglycosidase digestions. Our biochemical and structural data indicate that: (I) the cytosolic domain of NEP restricts the conformation of the SA domain because mutants not secreted in their full-length form are secreted in their truncated form; (2) alpha-helix-breaking residues are not a prerequisite for cleavage; (3) the presence, in close proximity to a putative signal peptidase cleavage site, of a polar sequence that maintains the alpha-helical structure of the SA domain is sufficient to promote cleavage. Furthermore pulse chase studies suggest that cleavage is performed in the ER by signal peptidase and indicate that cleavage is not a limiting step in the biosynthesis of the soluble form of the protein.

Full Text

The Full Text of this article is available as a PDF (473.2 KB).

Selected References

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

  1. Aubry M., Berteloot A., Beaumont A., Roques B. P., Crine P. The use of a monoclonal antibody for the rapid purification of kidney neutral endopeptidase ("enkephalinase") solubilized in octyl glucoside. Biochem Cell Biol. 1987 Apr;65(4):398–404. doi: 10.1139/o87-050. [DOI] [PubMed] [Google Scholar]
  2. Bird P., Gething M. J., Sambrook J. The functional efficiency of a mammalian signal peptide is directly related to its hydrophobicity. J Biol Chem. 1990 May 25;265(15):8420–8425. [PubMed] [Google Scholar]
  3. Burgess T. L., Craik C. S., Matsuuchi L., Kelly R. B. In vitro mutagenesis of trypsinogen: role of the amino terminus in intracellular protein targeting to secretory granules. J Cell Biol. 1987 Aug;105(2):659–668. doi: 10.1083/jcb.105.2.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chang C. T., Wu C. S., Yang J. T. Circular dichroic analysis of protein conformation: inclusion of the beta-turns. Anal Biochem. 1978 Nov;91(1):13–31. doi: 10.1016/0003-2697(78)90812-6. [DOI] [PubMed] [Google Scholar]
  5. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  6. Corbeil D., Boileau G., Lemay G., Crine P. Expression and polarized apical secretion in Madin-Darby canine kidney cells of a recombinant soluble form of neutral endopeptidase lacking the cytosolic and transmembrane domains. J Biol Chem. 1992 Feb 5;267(4):2798–2801. [PubMed] [Google Scholar]
  7. Deléage G., Clerc F. F., Roux B., Gautheron D. C. ANTHEPROT: a package for protein sequence analysis using a microcomputer. Comput Appl Biosci. 1988 Aug;4(3):351–356. doi: 10.1093/bioinformatics/4.3.351. [DOI] [PubMed] [Google Scholar]
  8. Devault A., Lazure C., Nault C., Le Moual H., Seidah N. G., Chrétien M., Kahn P., Powell J., Mallet J., Beaumont A. Amino acid sequence of rabbit kidney neutral endopeptidase 24.11 (enkephalinase) deduced from a complementary DNA. EMBO J. 1987 May;6(5):1317–1322. doi: 10.1002/j.1460-2075.1987.tb02370.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dion N., Le Moual H., Crine P., Boileau G. Kinetic evidence that His-711 of neutral endopeptidase 24.11 is involved in stabilization of the transition state. FEBS Lett. 1993 Mar 8;318(3):301–304. doi: 10.1016/0014-5793(93)80533-z. [DOI] [PubMed] [Google Scholar]
  10. Ehlers M. R., Riordan J. F. Membrane proteins with soluble counterparts: role of proteolysis in the release of transmembrane proteins. Biochemistry. 1991 Oct 22;30(42):10065–10074. doi: 10.1021/bi00106a001. [DOI] [PubMed] [Google Scholar]
  11. Fasman G. D. Distinguishing transmembrane helices from peripheral helices by circular dichrosim. Biotechnol Appl Biochem. 1993 Oct;18(Pt 2):111–138. [PubMed] [Google Scholar]
  12. Folz R. J., Gordon J. I. Deletion of the propeptide from human preproapolipoprotein A-II redirects cotranslational processing by signal peptidase. J Biol Chem. 1986 Nov 5;261(31):14752–14759. [PubMed] [Google Scholar]
  13. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  14. Hegner M., von Kieckebusch-Gück A., Falchetto R., James P., Semenza G., Mantei N. Single amino acid substitutions can convert the uncleaved signal-anchor of sucrase-isomaltase to a cleaved signal sequence. J Biol Chem. 1992 Aug 25;267(24):16928–16933. [PubMed] [Google Scholar]
  15. Heukeshoven J., Dernick R. Characterization of a solvent system for separation of water-insoluble poliovirus proteins by reversed-phase high-performance liquid chromatography. J Chromatogr. 1985 Jun 19;326:91–101. doi: 10.1016/s0021-9673(01)87434-3. [DOI] [PubMed] [Google Scholar]
  16. Hong W. J., Doyle D. Molecular dissection of the NH2-terminal signal/anchor sequence of rat dipeptidyl peptidase IV. J Cell Biol. 1990 Aug;111(2):323–328. doi: 10.1083/jcb.111.2.323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Howell S., Lanctôt C., Boileau G., Crine P. Expression of an enzymically active glycosylphosphatidylinositol-anchored form of neutral endopeptidase (EC 3.4.24.11) in Cos-1 cells. Biochem J. 1994 Apr 1;299(Pt 1):171–176. doi: 10.1042/bj2990171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Lemay G., Waksman G., Roques B. P., Crine P., Boileau G. Fusion of a cleavable signal peptide to the ectodomain of neutral endopeptidase (EC 3.4.24.11) results in the secretion of an active enzyme in COS-1 cells. J Biol Chem. 1989 Sep 15;264(26):15620–15623. [PubMed] [Google Scholar]
  21. Lemire I., Roy P., Boileau G. Translocation of neutral endopeptidase 24.11 mutants with deletions of the NH2-terminal cytosolic domain. Biochem Cell Biol. 1994 May-Jun;72(5-6):182–187. doi: 10.1139/o94-027. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Lippincott-Schwartz J., Yuan L., Tipper C., Amherdt M., Orci L., Klausner R. D. Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic. Cell. 1991 Nov 1;67(3):601–616. doi: 10.1016/0092-8674(91)90534-6. [DOI] [PubMed] [Google Scholar]
  24. Martoglio B., Hofmann M. W., Brunner J., Dobberstein B. The protein-conducting channel in the membrane of the endoplasmic reticulum is open laterally toward the lipid bilayer. Cell. 1995 Apr 21;81(2):207–214. doi: 10.1016/0092-8674(95)90330-5. [DOI] [PubMed] [Google Scholar]
  25. McKnight C. J., Briggs M. S., Gierasch L. M. Functional and nonfunctional LamB signal sequences can be distinguished by their biophysical properties. J Biol Chem. 1989 Oct 15;264(29):17293–17297. [PubMed] [Google Scholar]
  26. Murakami H., Masui H., Sato G. H., Sueoka N., Chow T. P., Kano-Sueoka T. Growth of hybridoma cells in serum-free medium: ethanolamine is an essential component. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1158–1162. doi: 10.1073/pnas.79.4.1158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nilsson I., Whitley P., von Heijne G. The COOH-terminal ends of internal signal and signal-anchor sequences are positioned differently in the ER translocase. J Cell Biol. 1994 Sep;126(5):1127–1132. doi: 10.1083/jcb.126.5.1127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Noel D., Nikaido K., Ames G. F. A single amino acid substitution in a histidine-transport protein drastically alters its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochemistry. 1979 Sep 18;18(19):4159–4165. doi: 10.1021/bi00586a017. [DOI] [PubMed] [Google Scholar]
  29. Nothwehr S. F., Gordon J. I. Eukaryotic signal peptide structure/function relationships. Identification of conformational features which influence the site and efficiency of co-translational proteolytic processing by site-directed mutagenesis of human pre(delta pro)apolipoprotein A-II. J Biol Chem. 1989 Mar 5;264(7):3979–3987. [PubMed] [Google Scholar]
  30. Paulson J. C., Weinstein J., Ujita E. L., Riggs K. J., Lai P. H. The membrane-binding domain of a rat liver Golgi sialyltransferase. Biochem Soc Trans. 1987 Aug;15(4):618–620. doi: 10.1042/bst0150618. [DOI] [PubMed] [Google Scholar]
  31. Perczel A., Park K., Fasman G. D. Analysis of the circular dichroism spectrum of proteins using the convex constraint algorithm: a practical guide. Anal Biochem. 1992 May 15;203(1):83–93. doi: 10.1016/0003-2697(92)90046-a. [DOI] [PubMed] [Google Scholar]
  32. Perlman D., Halvorson H. O. A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides. J Mol Biol. 1983 Jun 25;167(2):391–409. doi: 10.1016/s0022-2836(83)80341-6. [DOI] [PubMed] [Google Scholar]
  33. Roy P., Chatellard C., Lemay G., Crine P., Boileau G. Transformation of the signal peptide/membrane anchor domain of a type II transmembrane protein into a cleavable signal peptide. J Biol Chem. 1993 Feb 5;268(4):2699–2704. [PubMed] [Google Scholar]
  34. Schmid S. R., Spiess M. Deletion of the amino-terminal domain of asialoglycoprotein receptor H1 allows cleavage of the internal signal sequence. J Biol Chem. 1988 Nov 15;263(32):16886–16891. [PubMed] [Google Scholar]
  35. Strauss A. W., Zimmerman M., Boime I., Ashe B., Mumford R. A., Alberts A. W. Characterization of an endopeptidase involved in pre-protein processing. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4225–4229. doi: 10.1073/pnas.76.9.4225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. doi: 10.1073/pnas.84.14.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Virella G., Coelho I. M. Unexpected mobility of human lambda chains in sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Immunochemistry. 1974 Mar;11(3):157–160. doi: 10.1016/0019-2791(74)90213-4. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. Yamamoto Y., Taniyama Y., Kikuchi M. Important role of the proline residue in the signal sequence that directs the secretion of human lysozyme in Saccharomyces cerevisiae. Biochemistry. 1989 Mar 21;28(6):2728–2732. doi: 10.1021/bi00432a054. [DOI] [PubMed] [Google Scholar]
  42. Yang J. T., Wu C. S., Martinez H. M. Calculation of protein conformation from circular dichroism. Methods Enzymol. 1986;130:208–269. doi: 10.1016/0076-6879(86)30013-2. [DOI] [PubMed] [Google Scholar]
  43. Yang X. F., Chatellard C., Lazure C., Crine P., Boileau G. Insertion of hydrophilic amino acid residues in the signal peptide/membrane anchor domain of neprilysin (neutral endopeptidase-24.11) results in its cleavage: role of the position of insertion. Arch Biochem Biophys. 1994 Dec;315(2):382–386. doi: 10.1006/abbi.1994.1514. [DOI] [PubMed] [Google Scholar]
  44. de Jong W. W., Zweers A., Cohen L. H. Influence of single amino acid substitutions on electrophoretic mobility of sodium dodecyl sulfate-protein complexes. Biochem Biophys Res Commun. 1978 May 30;82(2):532–539. doi: 10.1016/0006-291x(78)90907-5. [DOI] [PubMed] [Google Scholar]
  45. von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 1986 Jun 11;14(11):4683–4690. doi: 10.1093/nar/14.11.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. von Heijne G. Patterns of amino acids near signal-sequence cleavage sites. Eur J Biochem. 1983 Jun 1;133(1):17–21. doi: 10.1111/j.1432-1033.1983.tb07424.x. [DOI] [PubMed] [Google Scholar]
  47. von Heijne G. The signal peptide. J Membr Biol. 1990 May;115(3):195–201. doi: 10.1007/BF01868635. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES