Mutational Analysis of the Properties of Caveolin-1

Abstract

Caveolin is a principal structural component of caveolae membranes in vivo Recently, a family of caveolin-related proteins has been identified; caveolin has been retermed caveolin-1. Caveolin family members share three characteristic properties: (i) detergent insolubility at low temperatures; (ii) self-oligomerization; and (iii) incorporation into low density Triton-insoluble fractions enriched in caveolae membranes. Here, we have used a deletion mutagenesis approach as a first step toward understanding which regions of caveolin-1 contribute to its unusual properties. Two caveolin-1 deletion mutants were created that lack either the C-terminal domain (Cav-1ΔC) or the N-terminal domain (Cav-1ΔN); these mutants were compared with the behavior of full-length caveolin-1 (Cav-1FL) expressed in parallel. Our results show that the N-terminal domain and membrane spanning segment are sufficient to form high molecular mass oligomers of caveolin-1. However, a complete caveolin-1 molecule is required for conveying detergent insolubility and incorporation into low density Triton-insoluble complexes. These data indicate that homo-oligomerization and an intact transmembrane are not sufficient to confer detergent insolubility, suggesting an unknown role for the C-terminal domain in this process. To better understand the role of the C-terminal domain, this region of caveolin-1 (residues 135-178) was expressed as a glutathione S-transferase fusion protein in Escherichia coli Purified recombinant glutathione S-transferase-C-Cav-1 was found to stably interact with full-length caveolin-1 but not with the two caveolin-1 deletion mutants. These results suggest that the C-terminal domain interacts with both the N-terminal and C-terminal domains of an adjacent caveolin-1 homo-oligomer. This appears to be a specific homo-typic interaction, because the C-terminal domain of caveolin-1 failed to interact with full-length forms of caveolin-2 and caveolin-3. Homo-typic interaction of the C-terminal domain with an adjacent homo-oligomer could provide a mechanism for clustering caveolin-1 homo-oligomers while excluding other caveolin family members. This type of lateral segregation event could promote caveolae membrane formation and contribute to the detergent insolubility of caveolins-1, −2, and −3. Caveolin is a principal structural component of caveolae membranes in vivo Recently, a family of caveolin-related proteins has been identified; caveolin has been retermed caveolin-1. Caveolin family members share three characteristic properties: (i) detergent insolubility at low temperatures; (ii) self-oligomerization; and (iii) incorporation into low density Triton-insoluble fractions enriched in caveolae membranes. Here, we have used a deletion mutagenesis approach as a first step toward understanding which regions of caveolin-1 contribute to its unusual properties. Two caveolin-1 deletion mutants were created that lack either the C-terminal domain (Cav-1ΔC) or the N-terminal domain (Cav-1ΔN); these mutants were compared with the behavior of full-length caveolin-1 (Cav-1FL) expressed in parallel. Our results show that the N-terminal domain and membrane spanning segment are sufficient to form high molecular mass oligomers of caveolin-1. However, a complete caveolin-1 molecule is required for conveying detergent insolubility and incorporation into low density Triton-insoluble complexes. These data indicate that homo-oligomerization and an intact transmembrane are not sufficient to confer detergent insolubility, suggesting an unknown role for the C-terminal domain in this process. To better understand the role of the C-terminal domain, this region of caveolin-1 (residues 135-178) was expressed as a glutathione S-transferase fusion protein in Escherichia coli Purified recombinant glutathione S-transferase-C-Cav-1 was found to stably interact with full-length caveolin-1 but not with the two caveolin-1 deletion mutants. These results suggest that the C-terminal domain interacts with both the N-terminal and C-terminal domains of an adjacent caveolin-1 homo-oligomer. This appears to be a specific homo-typic interaction, because the C-terminal domain of caveolin-1 failed to interact with full-length forms of caveolin-2 and caveolin-3. Homo-typic interaction of the C-terminal domain with an adjacent homo-oligomer could provide a mechanism for clustering caveolin-1 homo-oligomers while excluding other caveolin family members. This type of lateral segregation event could promote caveolae membrane formation and contribute to the detergent insolubility of caveolins-1, −2, and −3. INTRODUCTIONCaveolae are vesicular organelles located near or attached to the plasma membrane (1Yamada E. J. Biophys. Biochem. Cytol. 1955; 1: 445-458Crossref PubMed Scopus (519) Google Scholar, 2Severs N.J. J. Cell Sci. 1988; 90: 341-348Crossref PubMed Google Scholar). They represent an appendage of the plasma membrane. Caveolae are most abundant in endothelial cells, adipocytes, smooth muscle cells, and fibroblasts, although they are thought to exist in most cell types (reviewed in Refs. 3Lisanti M.P. Scherer P. Tang Z.-L. Sargiacomo M. Trends Cell Biol. 1994; 4: 231-235Abstract Full Text PDF PubMed Scopus (587) Google Scholar, 4Lisanti M.P. Tang Z.-T. Scherer P. Sargiacomo M. Methods Enzymol. 1995; 250: 655-668Crossref PubMed Scopus (117) Google Scholar, 5Lisanti M.P. Scherer P.E. Tang Z.-L. Kubler E. Koleske A.J. Sargiacomo M.S. Semin. Dev. Biol. 1995; 6: 47-58Crossref Scopus (31) Google Scholar). The exact function of caveolae remains largely unknown; however, they are thought to function in both cellular transport processes and signal transduction (3Lisanti M.P. Scherer P. Tang Z.-L. Sargiacomo M. Trends Cell Biol. 1994; 4: 231-235Abstract Full Text PDF PubMed Scopus (587) Google Scholar, 5Lisanti M.P. Scherer P.E. Tang Z.-L. Kubler E. Koleske A.J. Sargiacomo M.S. Semin. Dev. Biol. 1995; 6: 47-58Crossref Scopus (31) Google Scholar).Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes in vivo (6Glenney J.R. FEBS Lett. 1992; 314: 45-48Crossref PubMed Scopus (189) Google Scholar, 7Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y. Glenney J.R. Anderson R.G.W. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1853) Google Scholar). It has been proposed that caveolin functions as a scaffolding protein to organize and concentrate specific lipids (cholesterol and glyco-sphingolipids) (8Li S. Song K.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 568-573Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 9Murata M. Peranen J. Schreiner R. Weiland F. Kurzchalia T. Simons K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10339-10343Crossref PubMed Scopus (761) Google Scholar) and lipid-modified signaling molecules (Src-like kinases, H-Ras, and G-proteins) (10Li S. Okamoto T. Chun M. Sargiacomo M. Casanova J.E. Hansen S.H. Nishimoto I. Lisanti M.P. J. Biol. Chem. 1995; 270: 15693-15701Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar, 11Song K.S. Li S. Okamoto T. Quilliam L. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (916) Google Scholar, 12Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar) within caveolae micro-domains (13Sargiacomo M. Scherer P.E. Tang Z.-L. Kubler E. Song K.S. Sanders M.C. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9407-9411Crossref PubMed Scopus (475) Google Scholar). Recently, we and others have identified a family of caveolin-related proteins (caveolin-2 and caveolin-3) (14Scherer P.E. Okamoto T. Chun M. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 131-135Crossref PubMed Scopus (490) Google Scholar, 15Tang Z. Scherer P.E. Okamoto T. Song K. Chu C. Kohtz D.S. Nishimoto I. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1996; 271: 2255-2261Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar, 16Song K.S. Scherer P.E. Tang Z. Okamoto T. Li S. Chafel M. Chu C. Kohtz D.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 15160-15165Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar, 17Way M. Parton R. FEBS Lett. 1995; 376: 108-112Crossref PubMed Scopus (260) Google Scholar); caveolin has been retermed caveolin-1 (14Scherer P.E. Okamoto T. Chun M. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 131-135Crossref PubMed Scopus (490) Google Scholar).Caveolin-1 appears to be an essential component of caveolae (18Li S. Song K.S. Koh S. Kikuchi A. Lisanti M.P. J. Biol. Chem. 1996; 271: 28647-28654Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). For example, caveolin-1 protein expression directly parallels caveolae formation during adipocyte differentiation (14Scherer P.E. Okamoto T. Chun M. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 131-135Crossref PubMed Scopus (490) Google Scholar, 19Fan J.Y. Carpentier J.-L. van Obberghen E. Grunfeld C. Gorden P. Orci L. J. Cell Sci. 1983; 61: 219-230Crossref PubMed Google Scholar, 20Scherer P.E. Lisanti M.P. Baldini G. Sargiacomo M. Corley-Mastick C. Lodish H.F. J. Cell Biol. 1994; 127: 1233-1243Crossref PubMed Scopus (352) Google Scholar). Conversely, caveolin-1 mRNA and protein expression are lost or reduced during cell transformation, and caveolae are absent from these cell lines (21Koleske A.J. Baltimore D. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1381-1385Crossref PubMed Scopus (470) Google Scholar). Recombinant over-expression of caveolin-1 in caveolin-deficient cell lines results in: (i) the correct biochemical targeting of caveolin-1 to caveolae-enriched membrane fractions in FRT cells (22Scherer P.E. Tang Z. Chun M. Sargiacomo M. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1995; 270: 16395-16401Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar) and (ii) the formation of recombinant caveolae vesicles in lymphocytes (23Fra A.M. Williamson E. Simons K. Parton R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8655-8659Crossref PubMed Scopus (524) Google Scholar) and Sf21 insect cells (18Li S. Song K.S. Koh S. Kikuchi A. Lisanti M.P. J. Biol. Chem. 1996; 271: 28647-28654Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). These results provide direct evidence that caveolin family members participate in caveolae formation.However, it remains unknown how caveolin-1 expression induces caveolae formation. This may be related to the self-assembly properties of caveolin-1. Caveolin-1 undergoes two stages of oligomerization. First, in the endoplasmic reticulum, caveolin-1 monomers assemble into discrete multivalent homo-oligomers, containing ∼14-16 monomers per oligomer (13Sargiacomo M. Scherer P.E. Tang Z.-L. Kubler E. Song K.S. Sanders M.C. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9407-9411Crossref PubMed Scopus (475) Google Scholar, 24Monier S. Parton R.G. Vogel F. Behlke J. Henske A. Kurzchalia T. Mol. Biol. Cell. 1995; 6: 911-927Crossref PubMed Scopus (397) Google Scholar). Subsequently, these individual caveolin-1 homo-oligomers (4-6 nm spherical particles) can interact with each other to form clusters of particles that are ∼25-50 nm in diameter (13Sargiacomo M. Scherer P.E. Tang Z.-L. Kubler E. Song K.S. Sanders M.C. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9407-9411Crossref PubMed Scopus (475) Google Scholar). Also caveolin-1 homo-oligomers interact specifically with glycosphingolipids (25Fra A.M. Masserini M. Palestini P. Sonnino S. Simons K. FEBS Lett. 1995; 375: 11-14Crossref PubMed Scopus (160) Google Scholar) and cholesterol (8Li S. Song K.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 568-573Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 9Murata M. Peranen J. Schreiner R. Weiland F. Kurzchalia T. Simons K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10339-10343Crossref PubMed Scopus (761) Google Scholar) and require a high cholesterol content (≥30%) to insert into model lipid membranes (8Li S. Song K.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 568-573Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 9Murata M. Peranen J. Schreiner R. Weiland F. Kurzchalia T. Simons K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10339-10343Crossref PubMed Scopus (761) Google Scholar). Thus, we envisage that through the interaction of caveolin-1 with itself and the caveolin-mediated selection of endogenous lipid components, a caveolae-sized vesicle is generated (18Li S. Song K.S. Koh S. Kikuchi A. Lisanti M.P. J. Biol. Chem. 1996; 271: 28647-28654Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar).The specialized lipid composition of caveolae is thought to convey resistance of this membrane domain to detergent solubilization by Triton X-100 (at low temperatures) (20Scherer P.E. Lisanti M.P. Baldini G. Sargiacomo M. Corley-Mastick C. Lodish H.F. J. Cell Biol. 1994; 127: 1233-1243Crossref PubMed Scopus (352) Google Scholar, 26Sargiacomo M. Sudol M. Tang Z.-L. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (858) Google Scholar, 27Lisanti M.P. Tang Z.-L. Sargiacomo M. J. Cell Biol. 1993; 123: 595-604Crossref PubMed Scopus (159) Google Scholar, 28Lisanti M.P. Scherer P.E. Vidugiriene J. Tang Z.-L. HermanoskiVosatka A. Tu Y.-H. Cook R.F. Sargiacomo M. J. Cell Biol. 1994; 126: 111-126Crossref PubMed Scopus (809) Google Scholar, 29Chang W.J. Ying Y. Rothberg K. Hooper N. Turner A. Gambliel H. De Gunzburg J. Mumby S. Gilman A. Anderson R.G.W. J. Cell Biol. 1994; 126: 127-138Crossref PubMed Scopus (309) Google Scholar, 30Schroeder R. London E. Brown D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12130-12134Crossref PubMed Scopus (635) Google Scholar, 31Schnitzer J.E. Oh P. Jacobson B.S. Dvorak A.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1759-1763Crossref PubMed Scopus (230) Google Scholar). This property appears to be unique to caveolae membranes. For example, when intact cells were fixed in paraformaldehyde, extracted with Triton X-100, and then examined by electron microscopy, the insoluble membranes that remained were found to be caveolae (32Moldovan N. Heltianu C. Simionescu N. Simionescu M. Exp. Cell Res. 1995; 219: 309-313Crossref PubMed Scopus (32) Google Scholar). However, it is not known whether caveolin-1 contributes to the detergent insolubility of caveolae membranes.Caveolin proteins can be divided into three distinct regions: (i) a cytoplasmic N-terminal domain; (ii) an unusual 33-amino acid hydrophobic membrane spanning segment; and (iii) a cytoplasmic C-terminal domain (6Glenney J.R. FEBS Lett. 1992; 314: 45-48Crossref PubMed Scopus (189) Google Scholar, 14Scherer P.E. Okamoto T. Chun M. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 131-135Crossref PubMed Scopus (490) Google Scholar, 15Tang Z. Scherer P.E. Okamoto T. Song K. Chu C. Kohtz D.S. Nishimoto I. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1996; 271: 2255-2261Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar, 17Way M. Parton R. FEBS Lett. 1995; 376: 108-112Crossref PubMed Scopus (260) Google Scholar, 22Scherer P.E. Tang Z. Chun M. Sargiacomo M. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1995; 270: 16395-16401Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 33Glenney J.R. Soppet D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10517-10521Crossref PubMed Scopus (339) Google Scholar, 34Kurzchalia T. Dupree P. Parton R.G. Kellner R. Virta H. Lehnert M. Simons K. J. Cell Biol. 1992; 118: 1003-1014Crossref PubMed Scopus (462) Google Scholar, 35Tang Z.-L. Scherer P.E. Lisanti M.P. Gene (Amst.). 1994; 147: 299-300Crossref PubMed Scopus (55) Google Scholar). Here, we have employed a deletion mutagenesis approach to dissect which regions of caveolin-1 are required for its detergent insolubility, homo-oligomerization, and targeting to low density Triton-insoluble membrane fractions that are enriched in caveolae-membranes. Our results suggest a novel role for the C-terminal domain in mediating homo-typic caveolin-caveolin interactions between individual caveolin-1 oligomers. INTRODUCTIONCaveolae are vesicular organelles located near or attached to the plasma membrane (1Yamada E. J. Biophys. Biochem. Cytol. 1955; 1: 445-458Crossref PubMed Scopus (519) Google Scholar, 2Severs N.J. J. Cell Sci. 1988; 90: 341-348Crossref PubMed Google Scholar). They represent an appendage of the plasma membrane. Caveolae are most abundant in endothelial cells, adipocytes, smooth muscle cells, and fibroblasts, although they are thought to exist in most cell types (reviewed in Refs. 3Lisanti M.P. Scherer P. Tang Z.-L. Sargiacomo M. Trends Cell Biol. 1994; 4: 231-235Abstract Full Text PDF PubMed Scopus (587) Google Scholar, 4Lisanti M.P. Tang Z.-T. Scherer P. Sargiacomo M. Methods Enzymol. 1995; 250: 655-668Crossref PubMed Scopus (117) Google Scholar, 5Lisanti M.P. Scherer P.E. Tang Z.-L. Kubler E. Koleske A.J. Sargiacomo M.S. Semin. Dev. Biol. 1995; 6: 47-58Crossref Scopus (31) Google Scholar). The exact function of caveolae remains largely unknown; however, they are thought to function in both cellular transport processes and signal transduction (3Lisanti M.P. Scherer P. Tang Z.-L. Sargiacomo M. Trends Cell Biol. 1994; 4: 231-235Abstract Full Text PDF PubMed Scopus (587) Google Scholar, 5Lisanti M.P. Scherer P.E. Tang Z.-L. Kubler E. Koleske A.J. Sargiacomo M.S. Semin. Dev. Biol. 1995; 6: 47-58Crossref Scopus (31) Google Scholar).Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes in vivo (6Glenney J.R. FEBS Lett. 1992; 314: 45-48Crossref PubMed Scopus (189) Google Scholar, 7Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y. Glenney J.R. Anderson R.G.W. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1853) Google Scholar). It has been proposed that caveolin functions as a scaffolding protein to organize and concentrate specific lipids (cholesterol and glyco-sphingolipids) (8Li S. Song K.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 568-573Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 9Murata M. Peranen J. Schreiner R. Weiland F. Kurzchalia T. Simons K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10339-10343Crossref PubMed Scopus (761) Google Scholar) and lipid-modified signaling molecules (Src-like kinases, H-Ras, and G-proteins) (10Li S. Okamoto T. Chun M. Sargiacomo M. Casanova J.E. Hansen S.H. Nishimoto I. Lisanti M.P. J. Biol. Chem. 1995; 270: 15693-15701Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar, 11Song K.S. Li S. Okamoto T. Quilliam L. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (916) Google Scholar, 12Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar) within caveolae micro-domains (13Sargiacomo M. Scherer P.E. Tang Z.-L. Kubler E. Song K.S. Sanders M.C. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9407-9411Crossref PubMed Scopus (475) Google Scholar). Recently, we and others have identified a family of caveolin-related proteins (caveolin-2 and caveolin-3) (14Scherer P.E. Okamoto T. Chun M. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 131-135Crossref PubMed Scopus (490) Google Scholar, 15Tang Z. Scherer P.E. Okamoto T. Song K. Chu C. Kohtz D.S. Nishimoto I. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1996; 271: 2255-2261Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar, 16Song K.S. Scherer P.E. Tang Z. Okamoto T. Li S. Chafel M. Chu C. Kohtz D.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 15160-15165Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar, 17Way M. Parton R. FEBS Lett. 1995; 376: 108-112Crossref PubMed Scopus (260) Google Scholar); caveolin has been retermed caveolin-1 (14Scherer P.E. Okamoto T. Chun M. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 131-135Crossref PubMed Scopus (490) Google Scholar).Caveolin-1 appears to be an essential component of caveolae (18Li S. Song K.S. Koh S. Kikuchi A. Lisanti M.P. J. Biol. Chem. 1996; 271: 28647-28654Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). For example, caveolin-1 protein expression directly parallels caveolae formation during adipocyte differentiation (14Scherer P.E. Okamoto T. Chun M. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 131-135Crossref PubMed Scopus (490) Google Scholar, 19Fan J.Y. Carpentier J.-L. van Obberghen E. Grunfeld C. Gorden P. Orci L. J. Cell Sci. 1983; 61: 219-230Crossref PubMed Google Scholar, 20Scherer P.E. Lisanti M.P. Baldini G. Sargiacomo M. Corley-Mastick C. Lodish H.F. J. Cell Biol. 1994; 127: 1233-1243Crossref PubMed Scopus (352) Google Scholar). Conversely, caveolin-1 mRNA and protein expression are lost or reduced during cell transformation, and caveolae are absent from these cell lines (21Koleske A.J. Baltimore D. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1381-1385Crossref PubMed Scopus (470) Google Scholar). Recombinant over-expression of caveolin-1 in caveolin-deficient cell lines results in: (i) the correct biochemical targeting of caveolin-1 to caveolae-enriched membrane fractions in FRT cells (22Scherer P.E. Tang Z. Chun M. Sargiacomo M. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1995; 270: 16395-16401Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar) and (ii) the formation of recombinant caveolae vesicles in lymphocytes (23Fra A.M. Williamson E. Simons K. Parton R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8655-8659Crossref PubMed Scopus (524) Google Scholar) and Sf21 insect cells (18Li S. Song K.S. Koh S. Kikuchi A. Lisanti M.P. J. Biol. Chem. 1996; 271: 28647-28654Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). These results provide direct evidence that caveolin family members participate in caveolae formation.However, it remains unknown how caveolin-1 expression induces caveolae formation. This may be related to the self-assembly properties of caveolin-1. Caveolin-1 undergoes two stages of oligomerization. First, in the endoplasmic reticulum, caveolin-1 monomers assemble into discrete multivalent homo-oligomers, containing ∼14-16 monomers per oligomer (13Sargiacomo M. Scherer P.E. Tang Z.-L. Kubler E. Song K.S. Sanders M.C. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9407-9411Crossref PubMed Scopus (475) Google Scholar, 24Monier S. Parton R.G. Vogel F. Behlke J. Henske A. Kurzchalia T. Mol. Biol. Cell. 1995; 6: 911-927Crossref PubMed Scopus (397) Google Scholar). Subsequently, these individual caveolin-1 homo-oligomers (4-6 nm spherical particles) can interact with each other to form clusters of particles that are ∼25-50 nm in diameter (13Sargiacomo M. Scherer P.E. Tang Z.-L. Kubler E. Song K.S. Sanders M.C. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9407-9411Crossref PubMed Scopus (475) Google Scholar). Also caveolin-1 homo-oligomers interact specifically with glycosphingolipids (25Fra A.M. Masserini M. Palestini P. Sonnino S. Simons K. FEBS Lett. 1995; 375: 11-14Crossref PubMed Scopus (160) Google Scholar) and cholesterol (8Li S. Song K.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 568-573Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 9Murata M. Peranen J. Schreiner R. Weiland F. Kurzchalia T. Simons K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10339-10343Crossref PubMed Scopus (761) Google Scholar) and require a high cholesterol content (≥30%) to insert into model lipid membranes (8Li S. Song K.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 568-573Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 9Murata M. Peranen J. Schreiner R. Weiland F. Kurzchalia T. Simons K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10339-10343Crossref PubMed Scopus (761) Google Scholar). Thus, we envisage that through the interaction of caveolin-1 with itself and the caveolin-mediated selection of endogenous lipid components, a caveolae-sized vesicle is generated (18Li S. Song K.S. Koh S. Kikuchi A. Lisanti M.P. J. Biol. Chem. 1996; 271: 28647-28654Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar).The specialized lipid composition of caveolae is thought to convey resistance of this membrane domain to detergent solubilization by Triton X-100 (at low temperatures) (20Scherer P.E. Lisanti M.P. Baldini G. Sargiacomo M. Corley-Mastick C. Lodish H.F. J. Cell Biol. 1994; 127: 1233-1243Crossref PubMed Scopus (352) Google Scholar, 26Sargiacomo M. Sudol M. Tang Z.-L. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (858) Google Scholar, 27Lisanti M.P. Tang Z.-L. Sargiacomo M. J. Cell Biol. 1993; 123: 595-604Crossref PubMed Scopus (159) Google Scholar, 28Lisanti M.P. Scherer P.E. Vidugiriene J. Tang Z.-L. HermanoskiVosatka A. Tu Y.-H. Cook R.F. Sargiacomo M. J. Cell Biol. 1994; 126: 111-126Crossref PubMed Scopus (809) Google Scholar, 29Chang W.J. Ying Y. Rothberg K. Hooper N. Turner A. Gambliel H. De Gunzburg J. Mumby S. Gilman A. Anderson R.G.W. J. Cell Biol. 1994; 126: 127-138Crossref PubMed Scopus (309) Google Scholar, 30Schroeder R. London E. Brown D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12130-12134Crossref PubMed Scopus (635) Google Scholar, 31Schnitzer J.E. Oh P. Jacobson B.S. Dvorak A.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1759-1763Crossref PubMed Scopus (230) Google Scholar). This property appears to be unique to caveolae membranes. For example, when intact cells were fixed in paraformaldehyde, extracted with Triton X-100, and then examined by electron microscopy, the insoluble membranes that remained were found to be caveolae (32Moldovan N. Heltianu C. Simionescu N. Simionescu M. Exp. Cell Res. 1995; 219: 309-313Crossref PubMed Scopus (32) Google Scholar). However, it is not known whether caveolin-1 contributes to the detergent insolubility of caveolae membranes.Caveolin proteins can be divided into three distinct regions: (i) a cytoplasmic N-terminal domain; (ii) an unusual 33-amino acid hydrophobic membrane spanning segment; and (iii) a cytoplasmic C-terminal domain (6Glenney J.R. FEBS Lett. 1992; 314: 45-48Crossref PubMed Scopus (189) Google Scholar, 14Scherer P.E. Okamoto T. Chun M. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. 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