Flotillin and Epidermal Surface Antigen Define a New Family of Caveolae-associated Integral Membrane Proteins

Abstract

Caveolae are plasmalemmal microdomains that are involved in vesicular trafficking and signal transduction. We have sought to identify novel integral membrane proteins of caveolae. Here we describe the identification and molecular cloning of flotillin. By several independent methods, flotillin behaves as a resident integral membrane protein component of caveolae. Furthermore, we have identified epidermal surface antigen both as a flotillin homologue and as a resident caveolar protein. Significantly, flotillin is a marker for the Triton-insoluble, buoyant membrane fraction in brain, where to date mRNA species for known caveolin gene family members have not been detected. Caveolae are plasmalemmal microdomains that are involved in vesicular trafficking and signal transduction. We have sought to identify novel integral membrane proteins of caveolae. Here we describe the identification and molecular cloning of flotillin. By several independent methods, flotillin behaves as a resident integral membrane protein component of caveolae. Furthermore, we have identified epidermal surface antigen both as a flotillin homologue and as a resident caveolar protein. Significantly, flotillin is a marker for the Triton-insoluble, buoyant membrane fraction in brain, where to date mRNA species for known caveolin gene family members have not been detected. Within mammalian cells, proteins are segregated into distinct organellar membrane compartments, and this localization influences the function of these proteins. One such class of compartments is the set of plasmalemmal microdomains known as caveolae (1Yamada E. J. Biophys. Biochem. Cytol. 1955; 1: 445-458Google Scholar, 2Severs N.J. J. Cell Sci. 1988; 90: 341-348Google Scholar). These “little caves” are 50–100 nm invaginations of the plasma membrane that have distinct morphological and biochemical properties (3Anderson R.G.W. Curr. Opin. Cell Biol. 1993; 5: 647-652Google Scholar, 4Travis J. Science. 1993; 262: 1208-1209Google Scholar). Caveolae are present to some degree in most cell types, but they are particularly abundant in endothelial cells, adipocytes, fibroblasts, smooth muscle cells, and type I pneumocytes (5Lisanti M.P. Scherer P.E. Tang Z.-L. Sargiacomo M. Trends Cell Biol. 1994; 4: 231-235Google Scholar, 6Lisanti M.P. Scherer P.E. Tang Z.-L. Kubler E. Koleske A.J. Sargiacomo M.S. Semin. Dev. Biol. 1995; 6: 47-58Google Scholar). Coating the cytoplasmic surface of caveolae are concentric filaments composed at least in part by the family of 20–24-kDa integral membrane caveolin proteins (7Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y. Glenney J.R. Anderson R.G.W. Cell. 1992; 68: 673-682Google Scholar). In neurons plasma membrane invaginations that exhibit caveolae-like properties have been observed by electron microscopy (8Bouillot C. Prochiantz A. Rougon G. Allinquant B. J. Biol. Chem. 1996; 271: 7640-7644Google Scholar, 9Olive S. Dubois C. Schachner M. Rougon G. J. Neurochem. 1995; 65: 2307-2317Google Scholar), but mRNA species for known caveolin gene family members have not been detected. Caveolae have at least three functions (6Lisanti M.P. Scherer P.E. Tang Z.-L. Kubler E. Koleske A.J. Sargiacomo M.S. Semin. Dev. Biol. 1995; 6: 47-58Google Scholar). First, in endothelial cells, caveolae mediate the transcytosis of macromolecules from the vascular lumen to the sub-endothelial space (10Simionescu N. Simionescu M. Palade G.E. J. Cell Biol. 1975; 64: 586-607Google Scholar). In accord with this transport function, caveolae contain proteins that have been implicated in vesicular trafficking (11Schnitzer J.E. Liu J. Oh P. J. Biol. Chem. 1995; 270: 14399-14404Google Scholar). Moreover, in a cell-free system caveolae bud as vesicles from plasma membranes derived from endothelial cells in a time- and GTP-dependent manner, and in permeabilized cells GTP stimulates the endocytosis of cholera toxin B chain via caveolae (12Schnitzer J.E. Oh P. McIntosh D.P. Science. 1996; 274: 239-242Google Scholar). Second, caveolae are the sites of potocytosis, whereby small molecules are concentrated within caveolae by binding to glycosylphosphatidylinositol (GPI) 1The abbreviations used are: GPI, glycosylphosphatidylinositol; ESA, epidermal surface antigen; Mes, 4-morpholineethanesulfonic acid; PBS, phosphate-buffered saline; FCRD,flotillin cross-reactingdeterminant; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; ORF, open reading frame; P, plasma membrane; V, caveolae. 1The abbreviations used are: GPI, glycosylphosphatidylinositol; ESA, epidermal surface antigen; Mes, 4-morpholineethanesulfonic acid; PBS, phosphate-buffered saline; FCRD,flotillin cross-reactingdeterminant; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; ORF, open reading frame; P, plasma membrane; V, caveolae.-linked receptors and then traverse the plasma membrane into the cytoplasm via an unknown transporter (13Anderson R.G.W. Kamen B.A. Rothberg K.G. Lacey S.W. Science. 1992; 255: 410-411Google Scholar). Third, caveolae may participate in the relay of extracellular signals to the cell's interior by organizing signal transduction molecules (“the caveolae signaling hypothesis”) (5Lisanti M.P. Scherer P.E. Tang Z.-L. Sargiacomo M. Trends Cell Biol. 1994; 4: 231-235Google Scholar). Caveolin-rich membrane domains purified by either detergent-based or detergent-free methods are enriched in several distinct classes of signaling molecules, including Gα and Gβγ subunits of heterotrimeric GTP-binding proteins, Src-like tyrosine kinases, protein kinase Cα, and small GTP-binding proteins such as H-Ras and Rap GTPases (14Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Google Scholar, 15Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Google Scholar, 16Li S. Okamoto T. Chun M. Sargiacomo M. Casanova J.E. Hansen S.H. Nishimoto I. Lisanti M.P. J. Biol. Chem. 1995; 270: 15693-15701Google Scholar). Some of these signaling molecules physically interact with caveolin (15Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Google Scholar). During their biosynthesis, caveolin-1 monomers associate to form homo-oligomers of ∼350 kDa (14–16 monomers per oligomer) (17Sargiacomo 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: 9407-9411Google Scholar). These higher order structures of caveolin monomers may form a scaffold on which caveolin-interacting signaling molecules are organized or sequestered. These purified homo-oligomers also can undergo a second stage of oligomerization and assemble in vitro into structures that are similar in size to caveolae. However, it remains unknown whether other novel integral membrane proteins contribute to the structural organization of caveolae membranes in vivo. To systematically identify novel protein components of caveolae, we have purified membrane domains that are significantly enriched in caveolin from murine lung tissue (18Lisanti M.P. Tang Z.-T. Scherer P.E. Sargiacomo M. Methods Enzymol. 1995; 250: 655-668Google Scholar, 19Scherer P.E. Tang Z. Chun M. Sargiacomo M. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1995; 270: 16395-16401Google Scholar). This method of purification relies upon the characteristic insolubility of caveolae domains in Triton X-100 at 4 °C and their characteristic buoyant density when subjected to sucrose density gradient centrifugation. A ∼45-kDa component of these purified caveolin-rich membranes was one of 10 predominant polypeptides easily detected by Ponceau S staining (Fig. 1B in Ref. 19). Microsequencing of this ∼45-kDa component has revealed several novel peptide sequences, as well as peptide sequences from epidermal surface antigen (ESA). ESA was identified and cloned as a keratinocyte cell surface protein (20Schroeder W.T. Stewart-Galetka S. Mandavilli S. Parry D.A.D. Goldsmith L. Duvic M. J. Biol. Chem. 1994; 269: 19983-19991Google Scholar). Due to the emerging importance of caveolae in vesicular trafficking and signal transduction, we proceeded to clone the cDNA corresponding to the novel caveolae protein, which we have named “flotillin.” 2As we report, “flotillin” is a specific marker for the set of proteins that a of in the Triton-insoluble, buoyant fraction of from from from of the 2As we report, “flotillin” is a specific marker for the set of proteins that a of in the Triton-insoluble, buoyant fraction of from from from of the flotillin is a homologue of ESA, and they a family of integral membrane proteins in caveolae. tissue and was from a of and was from and to as J. Biol. Chem. Scholar). was from and from cells at and at of as G. T. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1992; Scholar), with the that used for of the was from the in 10 gel of to and of as G. T. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1992; Scholar). at °C in 10 and membranes in and at was at °C with an Microsequencing of the ∼45-kDa from lung caveolin-rich membrane domains was as P.E. Tang Z. Chun M. Sargiacomo M. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1995; 270: 16395-16401Google Scholar). and upon the novel peptide sequences in by These and used for with lung cDNA as the lung cDNA was from of lung to the A was by as used the and the where with the of and of in a as °C for °C for °C for °C for °C for °C for °C for and °C for 10 This not a this was used as for the second with and for of the second to the for the °C for °C for °C for was into to the and to to an open reading by sequences to the from the we that this cDNA to the protein from caveolin-rich membrane also from sequences from caveolin-rich membrane domains membrane domains purified from lung and to the protein and with then by and subjected to peptide as M.P. Scherer P.E. J. Tang Z.-L. A. Sargiacomo M. J. Cell Biol. 1994; Scholar). ∼45-kDa was of proteins one novel and one that to in the a that was by in a Caveolin-rich membrane domains purified from lung and to the protein and with then by and subjected to peptide as M.P. Scherer P.E. J. Tang Z.-L. A. Sargiacomo M. J. Cell Biol. 1994; Scholar). ∼45-kDa was of proteins one novel and one that to in the a that was by from at of was used as the to a to the was used to the cDNA to to the the cDNA was a gradient as A. B. Proc. Natl. Acad. Sci. U. S. A. Scholar). independent was and this was used for cDNA was with a cDNA that to the clone by was to the of which To a cDNA that to the of the cDNA was used as in with a and flotillin and of by the was then and used to the as A clone was the was from the via as in the This was then on both by S. and by an This clone to in the the we this by the of flotillin as from an from This an to that from the and with the the in from these and into cells by the method B. A. Proc. Natl. Acad. Sci. U. S. A. Scholar). cells for 10 in of and and with of cells with and at and at then for and with of cells with and then into Triton and by for 10 at cell then for at 4 °C with was by and was with the corresponding was for at 4 then with from the by in and by Scholar). gel was for by with for gel was to at a flotillin peptide in by This within was to and a to which the peptide been to methods E. A 1988; Scholar). a peptide in by as was from and from was as P.E. S. M. G. Lodish H.F. J. Biol. Chem. 1995; 270: Scholar). of with was to a Y. S. J. Cell Biol. Scholar). in and in a cells then into of and by in a at 4 the at at 4 °C in a in of and and with an of in the was by several a to the these then used for and of cells at of with PBS, in on for and then for 10 at 4 °C in a at on the the Caveolin-rich membrane domains purified to an method Cell. 1992; 68: Scholar, M.P. Scherer P.E. J. Tang Z.-L. A. Sargiacomo M. J. Cell Biol. 1994; Scholar, M. M. Tang Z.-L. Lisanti M.P. J. Cell Biol. 1993; with with PBS, into of Mes, with Triton and and by 10 in a was then to sucrose by of of an sucrose in to an and with of a sucrose gradient in and at the but Triton for at 4 °C in a at membranes as a at a density of sucrose (18Lisanti M.P. Tang Z.-T. Scherer P.E. Sargiacomo M. Methods Enzymol. 1995; 250: 655-668Google Scholar). of fraction by and was to a to that of the in and in a to Caveolin-rich membrane domains from purified by of a (14Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Google with in and then into of then on with a tissue on 10 for and a on for and then for was then to sucrose by of of sucrose in and at the of an A sucrose gradient that and was and at for in an purification of plasma membranes and caveolae was as J.E. McIntosh D.P. Liu J. Oh P. Science. 1995; 269: Scholar). proteins to a membranes in or with and and in or with and detected by to the of proteins by was to Scholar). of and protein sequences was with the J. Biol. Scholar), and 1994; Scholar). was with a and of a ∼45-kDa component of purified caveolin-rich membrane domains revealed several novel peptide sequences To clone the cDNA for this novel protein, we a by with that in with the the we then an cDNA A clone was that an open reading that a of with a molecular of most is in as the However, the at has a at and A or at M. J. Biol. Chem. Scholar). We have not which of these as the for the flotillin To that the cDNA clone a protein, a with the and at the with the was in In a with these cells, a protein was with a that the not cDNA for flotillin a protein of with a molecular of which the size of the ∼45-kDa protein that was from caveolin-rich membrane domains of and in of these domains 10 to may an signal peptide or a second to is 1994; tyrosine sites at and In flotillin sites for by protein kinase and and protein kinase A However, are of flotillin from to is to form an that may form a with other flotillin P. S. and B. for at flotillin with ESA, for which both and cDNA have been (20Schroeder W.T. Stewart-Galetka S. Mandavilli S. Parry D.A.D. Goldsmith L. Duvic M. J. Biol. Chem. 1994; 269: 19983-19991Google Scholar, M. W.T. P. Duvic M. 1995; (Fig. cDNA for ESA was cloned from a keratinocyte cDNA that was with a This been and was to epidermal cells in an Moreover, the of to to cell and this was by upon these ESA has been implicated in epidermal cell the of is flotillin and ESA at the the sites in the protein kinase at and the tyrosine at are in also with ESA a of that in the of the (Fig. of this remains to domains in the flotillin are not in such is from ESA the for ESA to flotillin second such and ESA in ESA that this to membrane also a flotillin and proteins from the (Fig. for the of flotillin and the protein. are flotillin and the of the in protein are to their corresponding in flotillin. In the of of the of flotillin is to that of and or However, such are detected flotillin and the of the flotillin was identified by with flotillin mRNA in that also that flotillin is at the mRNA in muscle and lung (Fig. these are similar to the of caveolin-1 P.E. Lisanti M.P. G. Sargiacomo M. C. Lodish H.F. J. Cell Biol. 1994; flotillin is at higher in to lung is of the also of in and not is that flotillin mRNA is easily in In is mRNA for known caveolin family in P.E. Lisanti M.P. G. Sargiacomo M. C. Lodish H.F. J. Cell Biol. 1994; Scholar, P.E. Okamoto T. Chun M. Nishimoto I. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1996; Scholar, Z. Scherer P.E. Okamoto T. Song C. Nishimoto I. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1996; 271: Scholar). ESA mRNA has been detected in that contain such as and in and and at in (20Schroeder W.T. Stewart-Galetka S. Mandavilli S. Parry D.A.D. Goldsmith L. Duvic M. J. Biol. Chem. 1994; 269: 19983-19991Google Scholar). To flotillin at the protein we an in within flotillin was upon surface and the of a protein in of caveolin-rich membrane domains from lung not and from (Fig. not this Furthermore, the not the protein in the of the peptide that was used for but this protein in the of We have not which flotillin is either to flotillin or to staining by upon the domains in the (Fig. we that flotillin associate with membrane proteins from membranes at but integral membrane proteins Y. S. J. Cell Biol. Scholar). at in and then subjected to flotillin was present in the membrane as was integral membrane protein, caveolin-1 (Fig. for family is a marker and was present in the the of a in ESA, ESA was detected in the with flotillin and These that flotillin and ESA as integral membrane proteins. We have that caveolin-1 mRNA and protein as well as the of caveolae per the of into P.E. Lisanti M.P. G. Sargiacomo M. C. Lodish H.F. J. Cell Biol. 1994; Scholar). the mRNA of both caveolin-1 and and 4 of P.E. Lisanti M.P. G. Sargiacomo M. C. Lodish H.F. J. Cell Biol. 1994; Scholar, P.E. Okamoto T. Chun M. Nishimoto I. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1996; Scholar). In the of of flotillin mRNA is (Fig. By at which the cells have been for flotillin mRNA has to mRNA then to a by to to by for flotillin then We have a similar in mRNA for on of E. P. E. M. P. and which may a to one or components of the used for from to the protein flotillin the of caveolin-1 and 4 flotillin protein per of protein in cell by is a from 4 to and from to A is observed for a protein. We not whether this is a protein, a of or a of flotillin. we have this protein is protein of kDa that is by the flotillin on a of In to flotillin and this ∼45-kDa protein on of of the ∼45-kDa protein is a protein that is by the but is to either flotillin or flotillin protein is ESA protein remains at per of protein in cell (Fig. To the of specific localization of flotillin and ESA in caveolae, several methods used to caveolae membranes from and endothelial In three methods, with was used to the of membrane First, proteins from an that caveolin-1 and other proteins from the of proteins and This is upon the insolubility of caveolae membranes in the Triton X-100 at 4 °C and their specific buoyant density in sucrose this we have that caveolin-1 is purified to cell In caveolin-1 is from proteins and for and plasma which in of these sucrose density P.E. Tang Z. Chun M. Sargiacomo M. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1995; 270: 16395-16401Google Scholar, M.P. Scherer P.E. J. Tang Z.-L. A. Sargiacomo M. J. Cell Biol. 1994; Scholar, M. M. Tang Z.-L. Lisanti M.P. J. Cell Biol. 1993; Scholar). from these from the and the proteins of fraction by to and with the flotillin and ESA and of A the of protein the gradient by Ponceau S ESA, and caveolin-1 and are concentrated within fraction which to caveolin-rich membrane domains that can as a in the gradient (Fig. A and Significantly, ESA, and caveolin-1 are from the of proteins, which within and the remains in and and not with we also used a detergent-free to caveolin-rich membrane domains as we have (14Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Google Scholar). This the Triton X-100 with to this caveolin is enriched in a as with the but in to the the glycosylphosphatidylinositol protein is detected in buoyant We this method to the of adipocytes, and ESA, and caveolin-1 (Fig. in the fraction that to we used a independent method for the purification of caveolae in a cell type to the membrane in which flotillin and ESA In this the lung vascular system is with J.E. McIntosh D.P. Liu J. Oh P. Science. 1995; 269: Scholar). These to the plasma membranes of endothelial cells and their density such that they can purified by and centrifugation. membrane caveolae on the of the membrane the These caveolae are then of the plasma by at 4 °C in the of the Triton caveolae are then purified from the plasma by sucrose gradient centrifugation. this method of the caveolin signal is present in caveolae, of the signals for the proteins and are present in this method can used to membrane domains as caveolae, which are enriched in the marker protein and plasma membrane domains that are enriched in proteins. of these three V, and that ESA, and caveolin-1 are enriched in V, the purified caveolae (Fig. and at of was signal for flotillin also in of is detected in the fraction and is from the these that flotillin and homologue ESA are resident integral membrane protein components of caveolae. flotillin mRNA is easily in by (Fig. mRNA species for known caveolin family members are This to the of whether flotillin is at the protein in whether it in the membrane To these tissue was subjected to the purification that we used for as is in adipocytes, flotillin is present in the in tissue not in cell type or flotillin is in However, flotillin is in the membrane of cells not which are derived from a This is not that neurons the flotillin protein. In this we have a gene family of integral membrane proteins that flotillin and of the that flotillin and ESA are resident components of caveolae. First, that are within the sequences of flotillin and ESA have been identified by of caveolin-rich membrane domains purified from lung Second, an an of flotillin a protein that with caveolin-1 in either detergent-based or detergent-free purification ESA with caveolin-1 in the Third, caveolae vesicles that have been purified from lung endothelial cell plasma membranes with contain of the plasma membrane flotillin and ESA proteins. and flotillin have similar tissue at the mRNA However, one to this is brain, where flotillin is easily but caveolin family members are We have that flotillin protein is in and that it in the Triton-insoluble, buoyant membrane that caveolae or caveolae-like structures are present in (8Bouillot C. Prochiantz A. Rougon G. Allinquant B. J. Biol. Chem. 1996; 271: 7640-7644Google Scholar, 9Olive S. Dubois C. Schachner M. Rougon G. J. Neurochem. 1995; 65: 2307-2317Google Scholar, P. J. 1996; Scholar). flotillin may a marker for these structures in membrane domains in are to caveolin-rich membrane domains in and other in we have flotillin also a of kDa that we have not of the of One is that is a of flotillin that is is that it is a of flotillin. may an flotillin homologue or a protein that a However, several of First, is present in the plasma membrane of lung endothelial cells and but it is from the caveolar fraction Second, is in Triton X-100 in not with in to of flotillin. These are with a in which caveolar flotillin is into and other with the that the this is then the of at the may to the identification of for caveolar localization and flotillin and ESA and their with caveolin-1 in that flotillin and ESA are members of the gene In of these ESA to an ESA is in cells as as adipocytes, and endothelial To that ESA and flotillin a gene we that flotillin known as and ESA known as members of the flotillin gene family is A of these is the identification of proteins of the that to flotillin. These to a and function for the flotillin gene Triton X-100 buoyant membrane domains have been in the E. Lisanti M.P. J. Biol. Chem. 1996; 271: Scholar), flotillin have been identified in the S. flotillin and ESA is their of protein the ESA protein is at as cells into adipocytes, the flotillin protein is at least mRNA and protein and mRNA as well as distinct caveolae, also significantly P.E. Lisanti M.P. G. Sargiacomo M. C. Lodish H.F. J. Cell Biol. 1994; Scholar, P.E. Okamoto T. Chun M. Nishimoto I. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1996; Scholar). of ESA protein as ESA may in a in and to caveolae or the of ESA per may is the flotillin mRNA and protein as detected by and mRNA are on and of but flotillin protein by an order of the Some other has the of the of flotillin mRNA or the of the flotillin protein or One is that flotillin protein is by the of caveolin protein by the of caveolae, the of which also this is the function or functions of the flotillin gene localization to caveolae some First, flotillin and ESA may contribute to the of caveolae. One for caveolin function that caveolin monomers assemble into homo-oligomers that to form a scaffold which signaling molecules can (14Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Google Scholar, 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: 9407-9411Google Scholar). caveolin scaffold then a for organizing these signaling molecules and in a and ESA may structural components of this scaffold or may form the of a distinct part of the caveolae the caveolar function for flotillin and ESA is that they participate in either as receptors or as and ESA a tyrosine but whether they are at this remains to into the membrane of these proteins to whether they are a novel class of ESA, in was cloned on the of a keratinocyte cell surface the function for ESA is a in cell This is upon the of the to cell when it is to flotillin also a in cell is an proteins known to mediate or have been to in or caveolae A. J. Cell Biol. 1995; or to interact with caveolin Cell. 1996; Scholar). is for the of caveolae in cell the functions of flotillin and ESA, it to such similar proteins are present in the One is that flotillin and ESA are flotillin and ESA similar functions in for distinct of caveolae. flotillin and ESA may to a In the molecular cloning of and the identification of and as components of caveolae by which to the and function of caveolae We and for of the and for and for in