Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1997 Sep 15;16(18):5501–5508. doi: 10.1093/emboj/16.18.5501

Interaction of influenza virus haemagglutinin with sphingolipid-cholesterol membrane domains via its transmembrane domain.

P Scheiffele 1, M G Roth 1, K Simons 1
PMCID: PMC1170182  PMID: 9312009

Abstract

Sphingolipid-cholesterol rafts are microdomains in biological membranes with liquid-ordered phase properties which are implicated in membrane traffic and signalling events. We have used influenza virus haemagglutinin (HA) as a model protein to analyse the interaction of transmembrane proteins with these microdomains. Here we demonstrate that raft association is an intrinsic property encoded in the protein. Mutant HA molecules with foreign transmembrane domain (TMD) sequences lose their ability to associate with the lipid microdomains, and mutations in the HA TMD reveal a requirement for hydrophobic residues in contact with the exoplasmic leaflet of the membrane. We also provide experimental evidence that cholesterol is critically required for association of proteins with lipid rafts. Our data suggest that the binding to specific membrane domains can be encoded in transmembrane proteins and that this information will be used for polarized sorting and signal transduction processes.

Full Text

The Full Text of this article is available as a PDF (301.0 KB).

Selected References

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

  1. Arreaza G., Brown D. A. Sorting and intracellular trafficking of a glycosylphosphatidylinositol-anchored protein and two hybrid transmembrane proteins with the same ectodomain in Madin-Darby canine kidney epithelial cells. J Biol Chem. 1995 Oct 6;270(40):23641–23647. doi: 10.1074/jbc.270.40.23641. [DOI] [PubMed] [Google Scholar]
  2. Bretscher M. S., Munro S. Cholesterol and the Golgi apparatus. Science. 1993 Sep 3;261(5126):1280–1281. doi: 10.1126/science.8362242. [DOI] [PubMed] [Google Scholar]
  3. Brown D. A., Crise B., Rose J. K. Mechanism of membrane anchoring affects polarized expression of two proteins in MDCK cells. Science. 1989 Sep 29;245(4925):1499–1501. doi: 10.1126/science.2571189. [DOI] [PubMed] [Google Scholar]
  4. Casey P. J. Protein lipidation in cell signaling. Science. 1995 Apr 14;268(5208):221–225. doi: 10.1126/science.7716512. [DOI] [PubMed] [Google Scholar]
  5. Fiedler K., Kobayashi T., Kurzchalia T. V., Simons K. Glycosphingolipid-enriched, detergent-insoluble complexes in protein sorting in epithelial cells. Biochemistry. 1993 Jun 29;32(25):6365–6373. doi: 10.1021/bi00076a009. [DOI] [PubMed] [Google Scholar]
  6. Fra A. M., Williamson E., Simons K., Parton R. G. Detergent-insoluble glycolipid microdomains in lymphocytes in the absence of caveolae. J Biol Chem. 1994 Dec 9;269(49):30745–30748. [PubMed] [Google Scholar]
  7. Gorodinsky A., Harris D. A. Glycolipid-anchored proteins in neuroblastoma cells form detergent-resistant complexes without caveolin. J Cell Biol. 1995 May;129(3):619–627. doi: 10.1083/jcb.129.3.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hanada K., Nishijima M., Akamatsu Y., Pagano R. E. Both sphingolipids and cholesterol participate in the detergent insolubility of alkaline phosphatase, a glycosylphosphatidylinositol-anchored protein, in mammalian membranes. J Biol Chem. 1995 Mar 17;270(11):6254–6260. doi: 10.1074/jbc.270.11.6254. [DOI] [PubMed] [Google Scholar]
  9. Hannan L. A., Edidin M. Traffic, polarity, and detergent solubility of a glycosylphosphatidylinositol-anchored protein after LDL-deprivation of MDCK cells. J Cell Biol. 1996 Jun;133(6):1265–1276. doi: 10.1083/jcb.133.6.1265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Harder T., Simons K. Caveolae, DIGs, and the dynamics of sphingolipid-cholesterol microdomains. Curr Opin Cell Biol. 1997 Aug;9(4):534–542. doi: 10.1016/s0955-0674(97)80030-0. [DOI] [PubMed] [Google Scholar]
  11. Hughey P. G., Compans R. W., Zebedee S. L., Lamb R. A. Expression of the influenza A virus M2 protein is restricted to apical surfaces of polarized epithelial cells. J Virol. 1992 Sep;66(9):5542–5552. doi: 10.1128/jvi.66.9.5542-5552.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Klein U., Gimpl G., Fahrenholz F. Alteration of the myometrial plasma membrane cholesterol content with beta-cyclodextrin modulates the binding affinity of the oxytocin receptor. Biochemistry. 1995 Oct 24;34(42):13784–13793. doi: 10.1021/bi00042a009. [DOI] [PubMed] [Google Scholar]
  13. Kundu A., Avalos R. T., Sanderson C. M., Nayak D. P. Transmembrane domain of influenza virus neuraminidase, a type II protein, possesses an apical sorting signal in polarized MDCK cells. J Virol. 1996 Sep;70(9):6508–6515. doi: 10.1128/jvi.70.9.6508-6515.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kurzchalia T. V., Dupree P., Parton R. G., Kellner R., Virta H., Lehnert M., Simons K. VIP21, a 21-kD membrane protein is an integral component of trans-Golgi-network-derived transport vesicles. J Cell Biol. 1992 Sep;118(5):1003–1014. doi: 10.1083/jcb.118.5.1003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lazarovits J., Naim H. Y., Rodriguez A. C., Wang R. H., Fire E., Bird C., Henis Y. I., Roth M. G. Endocytosis of chimeric influenza virus hemagglutinin proteins that lack a cytoplasmic recognition feature for coated pits. J Cell Biol. 1996 Jul;134(2):339–348. doi: 10.1083/jcb.134.2.339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lazarovits J., Shia S. P., Ktistakis N., Lee M. S., Bird C., Roth M. G. The effects of foreign transmembrane domains on the biosynthesis of the influenza virus hemagglutinin. J Biol Chem. 1990 Mar 15;265(8):4760–4767. [PubMed] [Google Scholar]
  17. Lisanti M. P., Caras I. W., Davitz M. A., Rodriguez-Boulan E. A glycophospholipid membrane anchor acts as an apical targeting signal in polarized epithelial cells. J Cell Biol. 1989 Nov;109(5):2145–2156. doi: 10.1083/jcb.109.5.2145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lisanti M. P., Scherer P. E., Tang Z., Sargiacomo M. Caveolae, caveolin and caveolin-rich membrane domains: a signalling hypothesis. Trends Cell Biol. 1994 Jul;4(7):231–235. doi: 10.1016/0962-8924(94)90114-7. [DOI] [PubMed] [Google Scholar]
  19. Machamer C. E., Grim M. G., Esquela A., Chung S. W., Rolls M., Ryan K., Swift A. M. Retention of a cis Golgi protein requires polar residues on one face of a predicted alpha-helix in the transmembrane domain. Mol Biol Cell. 1993 Jul;4(7):695–704. doi: 10.1091/mbc.4.7.695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Matlin K. S., Simons K. Reduced temperature prevents transfer of a membrane glycoprotein to the cell surface but does not prevent terminal glycosylation. Cell. 1983 Aug;34(1):233–243. doi: 10.1016/0092-8674(83)90154-x. [DOI] [PubMed] [Google Scholar]
  21. Matter K., Mellman I. Mechanisms of cell polarity: sorting and transport in epithelial cells. Curr Opin Cell Biol. 1994 Aug;6(4):545–554. doi: 10.1016/0955-0674(94)90075-2. [DOI] [PubMed] [Google Scholar]
  22. Monier S., Dietzen D. J., Hastings W. R., Lublin D. M., Kurzchalia T. V. Oligomerization of VIP21-caveolin in vitro is stabilized by long chain fatty acylation or cholesterol. FEBS Lett. 1996 Jun 17;388(2-3):143–149. doi: 10.1016/0014-5793(96)00519-4. [DOI] [PubMed] [Google Scholar]
  23. Monier S., Parton R. G., Vogel F., Behlke J., Henske A., Kurzchalia T. V. VIP21-caveolin, a membrane protein constituent of the caveolar coat, oligomerizes in vivo and in vitro. Mol Biol Cell. 1995 Jul;6(7):911–927. doi: 10.1091/mbc.6.7.911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mostov K. E., de Bruyn Kops A., Deitcher D. L. Deletion of the cytoplasmic domain of the polymeric immunoglobulin receptor prevents basolateral localization and endocytosis. Cell. 1986 Nov 7;47(3):359–364. doi: 10.1016/0092-8674(86)90592-1. [DOI] [PubMed] [Google Scholar]
  25. Murata M., Peränen J., Schreiner R., Wieland F., Kurzchalia T. V., Simons K. VIP21/caveolin is a cholesterol-binding protein. Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10339–10343. doi: 10.1073/pnas.92.22.10339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Müsch A., Xu H., Shields D., Rodriguez-Boulan E. Transport of vesicular stomatitis virus G protein to the cell surface is signal mediated in polarized and nonpolarized cells. J Cell Biol. 1996 May;133(3):543–558. doi: 10.1083/jcb.133.3.543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Naim H. Y., Amarneh B., Ktistakis N. T., Roth M. G. Effects of altering palmitylation sites on biosynthesis and function of the influenza virus hemagglutinin. J Virol. 1992 Dec;66(12):7585–7588. doi: 10.1128/jvi.66.12.7585-7588.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pimplikar S. W., Ikonen E., Simons K. Basolateral protein transport in streptolysin O-permeabilized MDCK cells. J Cell Biol. 1994 Jun;125(5):1025–1035. doi: 10.1083/jcb.125.5.1025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rodgers W., Crise B., Rose J. K. Signals determining protein tyrosine kinase and glycosyl-phosphatidylinositol-anchored protein targeting to a glycolipid-enriched membrane fraction. Mol Cell Biol. 1994 Aug;14(8):5384–5391. doi: 10.1128/mcb.14.8.5384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rodgers W., Rose J. K. Exclusion of CD45 inhibits activity of p56lck associated with glycolipid-enriched membrane domains. J Cell Biol. 1996 Dec;135(6 Pt 1):1515–1523. doi: 10.1083/jcb.135.6.1515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rodriguez-Boulan E., Powell S. K. Polarity of epithelial and neuronal cells. Annu Rev Cell Biol. 1992;8:395–427. doi: 10.1146/annurev.cb.08.110192.002143. [DOI] [PubMed] [Google Scholar]
  32. Rothberg K. G., Heuser J. E., Donzell W. C., Ying Y. S., Glenney J. R., Anderson R. G. Caveolin, a protein component of caveolae membrane coats. Cell. 1992 Feb 21;68(4):673–682. doi: 10.1016/0092-8674(92)90143-z. [DOI] [PubMed] [Google Scholar]
  33. Sarkar G., Sommer S. S. The "megaprimer" method of site-directed mutagenesis. Biotechniques. 1990 Apr;8(4):404–407. [PubMed] [Google Scholar]
  34. Sato M., Sato K., Nakano A. Endoplasmic reticulum localization of Sec12p is achieved by two mechanisms: Rer1p-dependent retrieval that requires the transmembrane domain and Rer1p-independent retention that involves the cytoplasmic domain. J Cell Biol. 1996 Jul;134(2):279–293. doi: 10.1083/jcb.134.2.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Scheiffele P., Peränen J., Simons K. N-glycans as apical sorting signals in epithelial cells. Nature. 1995 Nov 2;378(6552):96–98. doi: 10.1038/378096a0. [DOI] [PubMed] [Google Scholar]
  36. Schroeder R., London E., Brown D. Interactions between saturated acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)-anchored proteins: GPI-anchored proteins in liposomes and cells show similar behavior. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12130–12134. doi: 10.1073/pnas.91.25.12130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shenoy-Scaria A. M., Dietzen D. J., Kwong J., Link D. C., Lublin D. M. Cysteine3 of Src family protein tyrosine kinase determines palmitoylation and localization in caveolae. J Cell Biol. 1994 Jul;126(2):353–363. doi: 10.1083/jcb.126.2.353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Simons K., Ikonen E. Functional rafts in cell membranes. Nature. 1997 Jun 5;387(6633):569–572. doi: 10.1038/42408. [DOI] [PubMed] [Google Scholar]
  39. Singer S. J., Nicolson G. L. The fluid mosaic model of the structure of cell membranes. Science. 1972 Feb 18;175(4023):720–731. doi: 10.1126/science.175.4023.720. [DOI] [PubMed] [Google Scholar]
  40. Skibbens J. E., Roth M. G., Matlin K. S. Differential extractability of influenza virus hemagglutinin during intracellular transport in polarized epithelial cells and nonpolar fibroblasts. J Cell Biol. 1989 Mar;108(3):821–832. doi: 10.1083/jcb.108.3.821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Srinivas R. V., Balachandran N., Alonso-Caplen F. V., Compans R. W. Expression of herpes simplex virus glycoproteins in polarized epithelial cells. J Virol. 1986 May;58(2):689–693. doi: 10.1128/jvi.58.2.689-693.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stefanová I., Horejsí V., Ansotegui I. J., Knapp W., Stockinger H. GPI-anchored cell-surface molecules complexed to protein tyrosine kinases. Science. 1991 Nov 15;254(5034):1016–1019. doi: 10.1126/science.1719635. [DOI] [PubMed] [Google Scholar]
  43. Yancey P. G., Rodrigueza W. V., Kilsdonk E. P., Stoudt G. W., Johnson W. J., Phillips M. C., Rothblat G. H. Cellular cholesterol efflux mediated by cyclodextrins. Demonstration Of kinetic pools and mechanism of efflux. J Biol Chem. 1996 Jul 5;271(27):16026–16034. doi: 10.1074/jbc.271.27.16026. [DOI] [PubMed] [Google Scholar]
  44. Yoshimori T., Keller P., Roth M. G., Simons K. Different biosynthetic transport routes to the plasma membrane in BHK and CHO cells. J Cell Biol. 1996 Apr;133(2):247–256. doi: 10.1083/jcb.133.2.247. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES