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. 1996 Nov 1;319(Pt 3):887–896. doi: 10.1042/bj3190887

Isolation and characterization of two distinct low-density, Triton-insoluble, complexes from porcine lung membranes.

E T Parkin 1, A J Turner 1, N M Hooper 1
PMCID: PMC1217871  PMID: 8920995

Abstract

The Triton-insoluble complex from porcine lung membranes has been separated into two distinct subfractions visible as discrete light-scattering bands following buoyant density-gradient centrifugation in sucrose. Both of these detergent-insoluble complexes were enriched in the glycosyl-phosphatidylinositol (GPI)-anchored ectoenzymes alkaline phosphatase, aminopeptidase P and 5'-nucleotidase, and both complexes excluded the polypeptide-anchored ectoenzymes angiotensin-converting enzyme, dipeptidyl peptidase IV and aminopeptidases A and N. The GPI-anchored proteins in both complexes were susceptible to release by phosphatidylinositol-specific phospholipase C. Both complexes were also enriched in cholesterol and glycosphingolipids, and in caveolin/VIP21, although only the higher-density fraction was enriched in the plasmalemmal caveolar marker proteins Ca(2+)-ATPase and the inositol 1,4,5-trisphosphate receptor. Among the annexin family of proteins, annexins I and IV were absent from the two detergent-insoluble complexes, annexin V was present in both, and annexins II and VI were only enriched in the higher-density fraction. When the mental chelator EGTA was present in the isolation buffers, annexins II and VI dissociated from the higher-density detergent-insoluble complex and only a single light-scattering band was observed on the sucrose gradient, at the same position as for the lower-density complex. In contrast, in the presence of excess calcium only a single detergent-insoluble complex was isolated from the sucrose gradients, at an intermediate density. Thus the detergent-insoluble membrane complex can be subfractionated on the basis of what appears to be calcium-dependent, annexin-mediated, vesicle aggregation into two distinct populations, only one of which is enriched in plasmalemmal caveolar marker proteins.

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Selected References

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  1. Ali S. M., Geisow M. J., Burgoyne R. D. A role for calpactin in calcium-dependent exocytosis in adrenal chromaffin cells. Nature. 1989 Jul 27;340(6231):313–315. doi: 10.1038/340313a0. [DOI] [PubMed] [Google Scholar]
  2. Anderson R. G. Caveolae: where incoming and outgoing messengers meet. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):10909–10913. doi: 10.1073/pnas.90.23.10909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anderson R. G., Kamen B. A., Rothberg K. G., Lacey S. W. Potocytosis: sequestration and transport of small molecules by caveolae. Science. 1992 Jan 24;255(5043):410–411. doi: 10.1126/science.1310359. [DOI] [PubMed] [Google Scholar]
  4. Arreaza G., Melkonian K. A., LaFevre-Bernt M., Brown D. A. Triton X-100-resistant membrane complexes from cultured kidney epithelial cells contain the Src family protein tyrosine kinase p62yes. J Biol Chem. 1994 Jul 22;269(29):19123–19127. [PubMed] [Google Scholar]
  5. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
  6. Brown D. A., Rose J. K. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell. 1992 Feb 7;68(3):533–544. doi: 10.1016/0092-8674(92)90189-j. [DOI] [PubMed] [Google Scholar]
  7. Cao L. G., Fishkind D. J., Wang Y. L. Localization and dynamics of nonfilamentous actin in cultured cells. J Cell Biol. 1993 Oct;123(1):173–181. doi: 10.1083/jcb.123.1.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chang W. J., Ying Y. S., Rothberg K. G., Hooper N. M., Turner A. J., Gambliel H. A., De Gunzburg J., Mumby S. M., Gilman A. G., Anderson R. G. Purification and characterization of smooth muscle cell caveolae. J Cell Biol. 1994 Jul;126(1):127–138. doi: 10.1083/jcb.126.1.127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chun M., Liyanage U. K., Lisanti M. P., Lodish H. F. Signal transduction of a G protein-coupled receptor in caveolae: colocalization of endothelin and its receptor with caveolin. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11728–11732. doi: 10.1073/pnas.91.24.11728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cinek T., Horejsí V. The nature of large noncovalent complexes containing glycosyl-phosphatidylinositol-anchored membrane glycoproteins and protein tyrosine kinases. J Immunol. 1992 Oct 1;149(7):2262–2270. [PubMed] [Google Scholar]
  11. Creutz C. E. The annexins and exocytosis. Science. 1992 Nov 6;258(5084):924–931. doi: 10.1126/science.1439804. [DOI] [PubMed] [Google Scholar]
  12. Dupree P., Parton R. G., Raposo G., Kurzchalia T. V., Simons K. Caveolae and sorting in the trans-Golgi network of epithelial cells. EMBO J. 1993 Apr;12(4):1597–1605. doi: 10.1002/j.1460-2075.1993.tb05804.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fujimoto T. Calcium pump of the plasma membrane is localized in caveolae. J Cell Biol. 1993 Mar;120(5):1147–1157. doi: 10.1083/jcb.120.5.1147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fujimoto T., Nakade S., Miyawaki A., Mikoshiba K., Ogawa K. Localization of inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae. J Cell Biol. 1992 Dec;119(6):1507–1513. doi: 10.1083/jcb.119.6.1507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Glenney J. R., Jr, Soppet D. Sequence and expression of caveolin, a protein component of caveolae plasma membrane domains phosphorylated on tyrosine in Rous sarcoma virus-transformed fibroblasts. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10517–10521. doi: 10.1073/pnas.89.21.10517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Glenney J. R., Jr, Zokas L. Novel tyrosine kinase substrates from Rous sarcoma virus-transformed cells are present in the membrane skeleton. J Cell Biol. 1989 Jun;108(6):2401–2408. doi: 10.1083/jcb.108.6.2401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Harder T., Gerke V. The annexin II2p11(2) complex is the major protein component of the triton X-100-insoluble low-density fraction prepared from MDCK cells in the presence of Ca2+. Biochim Biophys Acta. 1994 Sep 29;1223(3):375–382. doi: 10.1016/0167-4889(94)90098-1. [DOI] [PubMed] [Google Scholar]
  19. Hooper N. M., Bashir A. Glycosyl-phosphatidylinositol-anchored membrane proteins can be distinguished from transmembrane polypeptide-anchored proteins by differential solubilization and temperature-induced phase separation in Triton X-114. Biochem J. 1991 Dec 15;280(Pt 3):745–751. doi: 10.1042/bj2800745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hooper N. M., Turner A. J. Ectoenzymes of the kidney microvillar membrane. Aminopeptidase P is anchored by a glycosyl-phosphatidylinositol moiety. FEBS Lett. 1988 Mar 14;229(2):340–344. doi: 10.1016/0014-5793(88)81152-9. [DOI] [PubMed] [Google Scholar]
  21. Hooper N. M., Turner A. J. Ectoenzymes of the kidney microvillar membrane. Differential solubilization by detergents can predict a glycosyl-phosphatidylinositol membrane anchor. Biochem J. 1988 Mar 15;250(3):865–869. doi: 10.1042/bj2500865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hooper N. M., Turner A. J. Isolation of two differentially glycosylated forms of peptidyl-dipeptidase A (angiotensin converting enzyme) from pig brain: a re-evaluation of their role in neuropeptide metabolism. Biochem J. 1987 Feb 1;241(3):625–633. doi: 10.1042/bj2410625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Izumi T., Shibata Y., Yamamoto T. Striped structures on the cytoplasmic surface membranes of the endothelial vesicles of the rat aorta revealed by quick-freeze, deep-etching replicas. Anat Rec. 1988 Mar;220(3):225–232. doi: 10.1002/ar.1092200302. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Kurzchalia T. V., Hartmann E., Dupree P. Guilty by insolubility--does a protein's detergent insolubility reflect a caveolar location? Trends Cell Biol. 1995 May;5(5):187–189. doi: 10.1016/s0962-8924(00)88990-4. [DOI] [PubMed] [Google Scholar]
  26. Lisanti M. P., Scherer P. E., Vidugiriene J., Tang Z., Hermanowski-Vosatka A., Tu Y. H., Cook R. F., Sargiacomo M. Characterization of caveolin-rich membrane domains isolated from an endothelial-rich source: implications for human disease. J Cell Biol. 1994 Jul;126(1):111–126. doi: 10.1083/jcb.126.1.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lisanti M. P., Tang Z., Scherer P. E., Sargiacomo M. Caveolae purification and glycosylphosphatidylinositol-linked protein sorting in polarized epithelia. Methods Enzymol. 1995;250:655–668. doi: 10.1016/0076-6879(95)50103-7. [DOI] [PubMed] [Google Scholar]
  28. Macala L. J., Yu R. K., Ando S. Analysis of brain lipids by high performance thin-layer chromatography and densitometry. J Lipid Res. 1983 Sep;24(9):1243–1250. [PubMed] [Google Scholar]
  29. Mayor S., Maxfield F. R. Insolubility and redistribution of GPI-anchored proteins at the cell surface after detergent treatment. Mol Biol Cell. 1995 Jul;6(7):929–944. doi: 10.1091/mbc.6.7.929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mayor S., Rothberg K. G., Maxfield F. R. Sequestration of GPI-anchored proteins in caveolae triggered by cross-linking. Science. 1994 Jun 24;264(5167):1948–1951. doi: 10.1126/science.7516582. [DOI] [PubMed] [Google Scholar]
  31. Olive S., Dubois C., Schachner M., Rougon G. The F3 neuronal glycosylphosphatidylinositol-linked molecule is localized to glycolipid-enriched membrane subdomains and interacts with L1 and fyn kinase in cerebellum. J Neurochem. 1995 Nov;65(5):2307–2317. doi: 10.1046/j.1471-4159.1995.65052307.x. [DOI] [PubMed] [Google Scholar]
  32. Parpal S., Gustavsson J., Strålfors P. Isolation of phosphooligosaccharide/phosphoinositol glycan from caveolae and cytosol of insulin-stimulated cells. J Cell Biol. 1995 Oct;131(1):125–135. doi: 10.1083/jcb.131.1.125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Parton R. G., Joggerst B., Simons K. Regulated internalization of caveolae. J Cell Biol. 1994 Dec;127(5):1199–1215. doi: 10.1083/jcb.127.5.1199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Parton R. G. Ultrastructural localization of gangliosides; GM1 is concentrated in caveolae. J Histochem Cytochem. 1994 Feb;42(2):155–166. doi: 10.1177/42.2.8288861. [DOI] [PubMed] [Google Scholar]
  35. Raynal P., Pollard H. B. Annexins: the problem of assessing the biological role for a gene family of multifunctional calcium- and phospholipid-binding proteins. Biochim Biophys Acta. 1994 Apr 5;1197(1):63–93. doi: 10.1016/0304-4157(94)90019-1. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Sargiacomo M., Sudol M., Tang Z., Lisanti M. P. Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells. J Cell Biol. 1993 Aug;122(4):789–807. doi: 10.1083/jcb.122.4.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schnitzer J. E., Liu J., Oh P. Endothelial caveolae have the molecular transport machinery for vesicle budding, docking, and fusion including VAMP, NSF, SNAP, annexins, and GTPases. J Biol Chem. 1995 Jun 16;270(24):14399–14404. doi: 10.1074/jbc.270.24.14399. [DOI] [PubMed] [Google Scholar]
  39. Schnitzer J. E., McIntosh D. P., Dvorak A. M., Liu J., Oh P. Separation of caveolae from associated microdomains of GPI-anchored proteins. Science. 1995 Sep 8;269(5229):1435–1439. doi: 10.1126/science.7660128. [DOI] [PubMed] [Google Scholar]
  40. Schnitzer J. E., Oh P., Jacobson B. S., Dvorak A. M. Caveolae from luminal plasmalemma of rat lung endothelium: microdomains enriched in caveolin, Ca(2+)-ATPase, and inositol trisphosphate receptor. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1759–1763. doi: 10.1073/pnas.92.5.1759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Simionescu N. Cellular aspects of transcapillary exchange. Physiol Rev. 1983 Oct;63(4):1536–1579. doi: 10.1152/physrev.1983.63.4.1536. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Smart E. J., Ying Y. S., Mineo C., Anderson R. G. A detergent-free method for purifying caveolae membrane from tissue culture cells. Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10104–10108. doi: 10.1073/pnas.92.22.10104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Stahl A., Mueller B. M. The urokinase-type plasminogen activator receptor, a GPI-linked protein, is localized in caveolae. J Cell Biol. 1995 Apr;129(2):335–344. doi: 10.1083/jcb.129.2.335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Ying Y. S., Anderson R. G., Rothberg K. G. Each caveola contains multiple glycosyl-phosphatidylinositol-anchored membrane proteins. Cold Spring Harb Symp Quant Biol. 1992;57:593–604. doi: 10.1101/sqb.1992.057.01.065. [DOI] [PubMed] [Google Scholar]

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