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

(Received for publication, January 24, 1997, and in revised form, February 27, 1997)

Perry E. Bickel abc, Philipp E. Scherer ad, Jan E. Schnitzer ef, Phil Oh e, Michael P. Lisanti ag and Harvey F. Lodish ai

From the a Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142-1479, the b Department of Medicine, Diabetes Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, the e Department of Pathology, Harvard Medical School and Beth Israel Hospital, Boston, Massachusetts 02215, and the i Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


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.


INTRODUCTION

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 (1, 2). These "little caves" are 50-100 nm invaginations of the plasma membrane that have distinct morphological and biochemical properties (3, 4). 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 (5, 6). 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 (7). In neurons plasma membrane invaginations that exhibit caveolae-like properties have been observed by electron microscopy (8, 9), but mRNA species for known caveolin gene family members have not been detected.

Caveolae have at least three functions (6). First, in endothelial cells, caveolae mediate the transcytosis of macromolecules from the vascular lumen to the sub-endothelial space (10). In accord with this transport function, caveolae contain proteins that have been implicated in vesicular trafficking (11). 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 (12). Second, caveolae are the sites of potocytosis, whereby small molecules are concentrated within caveolae by binding to glycosylphosphatidylinositol (GPI)1-linked receptors and then traverse the plasma membrane into the cytoplasm via an unknown transporter (13). Third, caveolae may participate in the relay of extracellular signals to the cell's interior by organizing signal transduction molecules ("the caveolae signaling hypothesis") (5). Caveolin-rich membrane domains purified by either detergent-based or detergent-free methods are enriched in several distinct classes of signaling molecules, including Galpha and Gbeta gamma subunits of heterotrimeric GTP-binding proteins, Src-like tyrosine kinases, protein kinase Calpha , and small GTP-binding proteins such as H-Ras and Rap GTPases (14-16). Some of these signaling molecules physically interact with caveolin (15).

During their biosynthesis, caveolin-1 monomers associate to form homo-oligomers of ~350 kDa (14-16 monomers per oligomer) (17). 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 (18, 19). 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 (20). 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."2 Surprisingly, flotillin is a close homologue of ESA, and together they define a new family of integral membrane proteins in caveolae.


EXPERIMENTAL PROCEDURES

Materials

Dulbecco's modified Eagle's tissue culture medium lacking methionine, cysteine, and glutamine was purchased from ICN Pharmaceuticals, Inc. The Express Protein Labeling Reagent, a mixture of [35S]methionine and -cysteine was purchased from DuPont NEN.

Cell Culture

3T3-L1 mouse fibroblasts (American Type Culture Collection, Rockville, MD) were propagated and differentiated to adipocytes as described (21).

RNA Isolation and Analysis

Poly(A)+ RNA was isolated from mouse tissues and from 3T3-L1 cells at preconfluence and at progressive stages of differentiation as described (22), with the exception that RNA used for construction of the adipocyte phage library was eluted from the oligo(dT)-cellulose column in 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, without SDS. Formaldehyde/agarose gel electrophoresis of poly(A)+ RNA, transfer to nylon membranes, and 32P labeling of DNA were performed as described (22). Hybridizations were carried out overnight at 42 °C in 50% formamide, 5 × SSC, 25 mM sodium phosphate, pH 7.0, 10 × Denhardt's solution, 5 mM EDTA, 1% SDS, and 0.1 mg/ml poly(A). The nylon membranes were washed sequentially in 2 × SSC, 0.1% SDS, and 0.1 × SSC, 0.1% SDS at 50 °C. Autoradiography was performed at -80 °C with an intensifying screen.

Degenerate PCR Cloning of Flotillin

Microsequencing of the ~45-kDa region from mouse lung caveolin-rich membrane domains was performed as described (19). Sense and antisense degenerate primers were designed based upon the novel peptide sequences listed in Table I and were synthesized by Research Genetics, Huntsville, AL. These sense and antisense primer pairs were used for nested PCR with mouse lung first-strand cDNA as the template. Mouse lung first-strand cDNA was prepared from 1 µg of mouse lung polyadenylated RNA, according to the manufacturer's instructions (Life Technologies, Inc.). A discrete 0.3-kilobase pair band was generated by hemi-nested PCR as follows. The first reaction used the sense primer "DLV" (GAYHTNGTNAAYATGGGNAT) and the antisense primer "AYQ" (TGRTANGCNARRTCNGCYTG) (note: where Y = C,T; H = A,C,T; N = G,A,C,T; R = G,A; D = G,A,T) with the following conditions: 1.5 mM MgCl2, 0.2 mM each dNTP, 100 pmol of each primer, and 2.5 units of Amplitaq (Perkins-Elmer) in a 50-µl reaction volume. The cycling parameters were as follows: 95 °C for 3 min, (95 °C for 1 min, 42 °C for 1 min, 72 °C for 2 min) times 5 cycles, (95 °C for 1 min, 50 °C for 1 min, 72 °C for 2 min) times 25 cycles, and 72 °C for 10 min. This first reaction did not produce a discrete band. Of this first reaction 1 µl was used as template for the hemi-nested second reaction with sense primer DLV and antisense primer "EVN" (RTTNACYTCDATRTCRTA) (see note above for explanation). The conditions of the second reaction were identical to the first, except for the amplification cycles (95 °C for 1 min, 55 °C for 1.5 min, 72 °C for 2 min times 30 cycles). The 0.3-kilobase pair PCR product was subcloned into pCR-Script (Stratagene, Inc.) according to the manufacturer's instructions, sequenced, and found to correspond to an open reading frame. Because amino acid residues encoded by DNA sequences internal to the PCR primers matched residues from the microsequenced peptides, we concluded that this cDNA corresponded to the protein originally isolated from caveolin-rich membrane domains. The identical fragment could also be amplified from 3T3-L1 adipocyte first-strand cDNA.

Table I. Peptide sequences from caveolin-rich membrane domains (~45 kDa)

Caveolin-rich membrane domains were purified from mouse lung tissue. After SDS-PAGE and transfer to nitrocellulose, the protein bands were excised and digested with Lys-C. The resulting peptides were then separated by HPLC and subjected to peptide sequencing, as described (30). The ~45-kDa region was of particular interest because two proteins were identified, one novel and one that corresponded to ESA. An X in the sequence indicates a residue that was indeterminate by microsequencing. Caveolin-rich membrane domains were purified from mouse lung tissue. After SDS-PAGE and transfer to nitrocellulose, the protein bands were excised and digested with Lys-C. The resulting peptides were then separated by HPLC and subjected to peptide sequencing, as described (30). The ~45-kDa region was of particular interest because two proteins were identified, one novel and one that corresponded to ESA. An X in the sequence indicates a residue that was indeterminate by microsequencing.

Sequence Identity Residues

KVASSDLVNMGIXVVXXTLK Novel (flotillin) 133 -152
KXXYDIEVNXXXAQADLAYQL Novel (flotillin) 220 -240
KXQLIMQAXA Novel (flotillin) 302 -311
KXAFXEEVN ESA 174 -182
KXAEAQLAYELQGAXEQQK ESA 184 -202

Construction of a 3T3-L1 Adipocyte, Directional, Phage cDNA Library

Poly(A)+ RNA (5 µg) from 3T3-L1 adipocytes at day 8 of differentiation was used as the template to construct a lambda EXloxTM library according to the manufacturer's instructions (Novagen, Inc., Madison, WI). Oligo(dT) was used to prime the first-strand cDNA synthesis. Prior to ligation to the phage arms, the cDNA was size-fractionated (>1.5 kilobase pairs) using a potassium acetate gradient (5-20%) as described (23). The resulting library represented ~1 × 106 independent clones. The library was amplified once, and this singly amplified library was used for screening.

Isolation of a Full-Length Flotillin cDNA Clone

The 3T3-L1 adipocyte cDNA library described above was screened with a digoxigenin-labeled cDNA probe that corresponded to the original, partial clone generated by PCR (nucleotide position 571-843, Fig. 1). Screening was performed according to the manufacturer's instructions (Boehringer Mannheim). Several positive clones were obtained, none of which extended further 5' than base 296. To generate a cDNA probe that would correspond to the 5'-end of flotillin, the 3T3-L1 adipocyte cDNA library described above was used as template in nested PCR with a vector-based primer (T7 Gene-10: TGAGGTTGTAGAAGTTCCG) and two antisense flotillin primers (TGGTCATCATGAATATCC and AGCATCTCCTTGTTCTGG). After removal of vector sequence by restriction digestion, the resulting PCR fragment was then digoxigenin-labeled and used to screen the 3T3-L1 adipocyte library as above. A positive clone was plaque-purified. The plasmid containing the positive clone, pFlotillin-1, was excised from the phage arms via Cre-lox recombination as described in the manufacturer's protocol (Novagen, Inc.). This plasmid was then fully sequenced on both strands, by manual sequencing using Sequenase (U. S. Biochemical Corp.) and by automated sequencing using an Applied Biosystems 373 Sequencer. This clone extended to nucleotide position 146 in the 5'-untranslated region. Because the 5' PCR-generated fragment extended farther upstream than pFlotillin-1, we confirmed this upstream sequence by amplifying the 5'-end of flotillin as above from an independently constructed library (random-primed, mouse brain lambda gt10 library from CLONTECH). This fragment contained an identical sequence to that amplified from the adipocyte library, and even began with the same base. Accordingly, the sequence represented in Fig. 1 contains sequence data from these PCR-generated 5'-ends and pFlotillin-1.


Fig. 1. Nucleotide and predicted amino acid sequences of flotillin. Two potential transmembrane domains are boxed. The 14-amino acid peptide used to immunize rabbits for the production of the anti-flotillin polyclonal antisera is enclosed by an oval. Amino acid residues identified by microsequencing are underlined. The shaded box indicates the polyadenylation signal "ATTAAA" in the 3'-untranslated region. These cDNA sequence data are available from GenBank/EMBL under accession number U90435[GenBank].
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COS-7 Cell Transfections, in Vivo Labeling, and Immunoprecipitation

cDNAs were transiently transfected into COS-7 cells (10-cm dishes) by the DEAE-dextran method (24). 48 h after transfection, cells were labeled for 10 min in 3 ml of Dulbecco's modified Eagle's medium lacking methionine and cysteine and supplemented with 0.5 mCi (1000 Ci/mmol) of Express Protein Labeling Reagent. Thereafter, cells were washed 3 times with chase medium (Dulbecco's modified Eagle's medium containing unlabeled methionine and cysteine at 1 mM and cycloheximide at 300 µM). Cells were then incubated for 5, 10, 20, 30, and 60 min with 2 ml of chase medium. The cells were washed twice with cold PBS and then scraped into TNET/OG lysis buffer (1% Triton X-100, 60 mM octyl glucoside, 5 mM EDTA, 20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml leupeptin). Insoluble debris were removed by centrifugation for 10 min at 15,000 × g. The postnuclear cell lysates were then incubated for 30 min at 4 °C with Protein A-Sepharose (Pharmacia Biotech Inc.). The Protein A-Sepharose was removed by centrifugation, and fresh Protein A-Sepharose was added along with the corresponding antibody. Immunoprecipitation was performed for 3 h at 4 °C. Immunoprecipitates were then washed 5 times with lysis buffer (lacking octyl glucoside), released from the Protein A-Sepharose by boiling in Laemmli sample buffer, and analyzed by SDS-PAGE (25). The gel was prepared for fluorography by incubation with 1 M sodium salicylate for 20 min. The dried gel was exposed to Kodak X-OMAT AR film at -80 °C.

Antibodies

Antibodies against a flotillin peptide were generated in rabbits by Research Genetics, Huntsville, AL. This peptide, KAQRDYELKKATYD (residues 211-224, indicated within Fig. 1), was coupled to keyhole limpet hemocyanin and injected. The resulting antisera were affinity-purified over a CNBr-activated Sepharose column (Pharmacia) to which the immune peptide had been coupled according to standard methods (26). Antibodies against a carboxyl-terminal mouse GDI-1 peptide (FENMRKKQNDVFGEADQ) were generated in rabbits by Research Genetics as described above. Anti-Myc epitope IgG (mAb 9E10) was obtained from Santa Cruz Biotech. Anti-caveolin-1 IgG (mAb 2297) and anti-ESA monoclonal antibody were obtained from Transduction Laboratories. Polyclonal anti-Acrp30 antiserum was prepared as described (27).

Extraction of Cells with Sodium Carbonate

Extraction of 3T3-L1 adipocytes with sodium carbonate was performed according to a previously described protocol (28). Adipocytes were washed 3 times in PBS and once in a solution containing 150 mM NaCl. The cells were then scraped into 1 ml of 100 mM sodium bicarbonate buffer, pH 11.5, and homogenized by 5 strokes in a 2-ml Dounce homogenizer. After 30 min incubation at 4 °C, the homogenates were centrifuged at 100,000 rpm at 4 °C in a TLA 100.2 rotor (Beckman). The pellets were resuspended in 0.5 ml of 100 mM sodium bicarbonate buffer, pH 11.5, and pellet and supernatant fractions were immediately mixed with an equal volume of SDS-PAGE (Laemmli) sample buffer. Any DNA in the pellets was sheared by several passages through a 26-gauge needle to decrease the viscosity; these samples were then used directly for SDS-PAGE and Western blot analysis.

Preparation of Cell Lysates from 3T3-L1 Cells During Adipocyte Differentiation

Plates of 3T3-L1 cells at interval stages of adipogenesis were washed twice with cold PBS, lysed in TNET/OG lysis buffer, incubated on ice for 15 min, and then centrifuged for 10 min at 4 °C in a Beckman Microfuge at 14,000 rpm. Protein determinations were made on the post-nuclear supernatants using the BCA assay (Pierce).

Purification of Caveolin-rich Membrane Domains

Caveolin-rich membrane domains were purified according to an established method (29-31) with minor modifications. Briefly, 3T3-L1 adipocytes were washed 3 times with ice-cold PBS, scraped into 2 ml of Mes-buffered saline (25 mM Mes, pH 6.5, 150 mM NaCl; MBS) with 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml leupeptin and homogenized by 10 strokes in a glass Dounce homogenizer. The extract was then adjusted to 40% sucrose by addition of 2 ml of an 80% sucrose solution in MBS, transferred to an ultracentrifuge tube, and overlaid with 8 ml of a linear 5-30% sucrose gradient in MBS containing phenylmethylsulfonyl fluoride and leupeptin at the above concentrations but lacking Triton X-100. The gradients were centrifuged for 16 h at 4 °C in a Beckman SW41 rotor at 39,000 rpm. Caveolin-enriched membranes fractionate as a sharp, light-scattering band at a density of ~15-20% sucrose (18). Equal volumes of each fraction were analyzed by SDS-PAGE and Western blotting. The pellet was brought to a volume equal to that of the fractions in Laemmli sample buffer and solubilized in a Dounce homogenizer prior to boiling.

Detergent-free Purification of Caveolin-rich Membrane Domains

Caveolin-rich membrane domains from 3T3-L1 adipocytes were purified by means of a previously reported protocol (14) with minor modifications. 3T3-L1 adipocytes were washed 3 times in PBS and then scraped into 2 ml of 500 mM sodium carbonate, pH 11.0. Cells were then homogenized on ice with (i) a Polytron tissue grinder on output setting 10 for 30 s (Kinematica GmbH, Brinkmann Instruments, Westbury, NY) and (ii) a sonicator on output setting 2 for 30 s and then setting 5 for 5 bursts (Branson Sonifier 250, Branson Ultrasonic Corp., Danbury, CT). The homogenate was then adjusted to 45% sucrose by addition of 2 ml of 90% sucrose prepared in MBS and placed at the bottom of an ultracentrifuge tube. A 5-35% discontinuous sucrose gradient that contained MBS and 250 mM sodium carbonate was formed above and centrifuged at 39,000 rpm for 16 h in an SW41 rotor (Beckman Instruments, Palo Alto, CA).

Purification of Endothelial Plasma Membranes and Caveolae Using Cationic Colloidal Silica

Silica-based purification of plasma membranes and caveolae was performed as described (32).

Immunoblotting

After SDS-PAGE, proteins were transferred to BA83 nitrocellulose (Schleicher & Schuell) using a Hoefer Semi-Phor apparatus. Nitrocellulose membranes were blocked in PBS or Tris-buffered saline with 0.1% Tween 20 and 5% non-fat dry milk. Primary and secondary antibodies were diluted in PBS or Tris-buffered saline with 0.1% Tween 20 and 1% bovine serum albumin. Bound antibodies were detected by enhanced chemiluminescence according to the manufacturer's instructions (DuPont NEN).

Other Methods

Separation of proteins by SDS-PAGE (Laemmli) was performed according to standard protocols (25). Computer analysis of DNA and protein sequences was performed with the DNASTAR software package, BLAST search software (33), and MacPattern software (34). Densitometry was performed with a Bio-Rad model GS-700 Imaging Densitometer and Molecular Analyst Version 2.1.1 software (Bio-Rad).


RESULTS

Molecular Cloning of Flotillin, A Novel Caveolae-associated Protein

Microsequence analysis of a ~45-kDa component of purified caveolin-rich membrane domains revealed several novel peptide sequences (Table I). To clone the cDNA for this novel protein, we generated a probe by reverse transcription-PCR with degenerate primers that were designed in accordance with the microsequence data. With the resulting probe we then screened an appropriate full-length cDNA library. A ~1.7-kilobase clone was isolated that contained an open reading frame that predicted a polypeptide of 428 amino acids with a molecular mass of 47 kDa. The most 5'-encoded methionine is indicated in Fig. 1 as the initiator methionine. However, the next methionine at amino acid position 11 has a more classic Kozak consensus sequence (G at position +4 and A or G at position -3) (35). We have not yet defined experimentally which of these two ATG triplets serves as the start codon for the flotillin polypeptide.

To show that the isolated cDNA clone could encode a protein, a PCR-generated construct starting with the latter methionine and tagged at the carboxyl terminus with the Myc epitope (pMVVmyc) was expressed in COS-7 cells. In a metabolic pulse-chase experiment with these transfected cells, a ~50-kDa protein was immunoprecipitated with 9E10, a monoclonal antibody that recognizes the Myc epitope (data not shown).

Structural and Motif Analysis of Flotillin: Homology with ESA and Two ORFs in the Cyanobacterium Synechococcus

The cDNA for flotillin encodes a protein of 428 amino acids with a predicted molecular mass of 47 kDa, which closely matches the size of the ~45-kDa protein that was microsequenced from caveolin-rich membrane domains. Flotillin contains two hydrophobic domains of 27 and 18 amino acids, respectively (boxed regions in Fig. 1). The first of these domains (from position 10 to 36) may represent an atypical signal peptide or perhaps a transmembrane domain. The second hydrophobic region (from position 134 to 151) is another potential transmembrane domain. Computer-assisted motif analysis (34) reveals two potential tyrosine phosphorylation sites at positions 160 and 292. In addition, flotillin contains predicted sites for phosphorylation by protein kinase C (at positions 52, 150, 229, and 315) and protein kinase A (at position 222). However, there are no predicted N-linked glycosylation sites. The region of flotillin from 328 to 355 is predicted to form an alpha  helix that may form a triple coiled coil with other flotillin monomers.3

Beginning at position 47, flotillin demonstrates significant homology with ESA, for which both human and mouse cDNA clones have been reported (20, 36) (Fig. 2A). The cDNA for ESA was cloned from a human foreskin keratinocyte lambda gt11 cDNA library that was screened with a monoclonal antibody. This monoclonal antibody, ECS-1, had been raised against cultured human keratinocytes and was found to stain nucleated epidermal cells in an intercellular pattern. Moreover, the addition of ECS-1 to cultured mouse keratinocytes led to cell detachment, and this detachment was enhanced by complement. Based upon these data, ESA has been implicated in epidermal cell adhesion. Over the region of homology, there is ~47% identity between flotillin and ESA at the amino acid level. Of the potential phosphorylation sites in flotillin, only the protein kinase C phosphorylation site at residue 150 and the tyrosine phosphorylation site at residue 152 are conserved in ESA. Flotillin also shares with ESA a repeat of A/G, E, A/G, E that recurs 6 times in the carboxyl half of the polypeptide (Fig. 2A, shaded boxes). The significance of this repeat remains to be determined. The two potential membrane-spanning domains indicated in the flotillin sequence are not conserved in ESA. The first such domain (flotillin residues 10-36) is missing entirely from ESA because the initiator methionine for ESA corresponds to flotillin residue 46. The second such domain (flotillin residues 134-151 and ESA residues 89-106) contains two additional charged amino acids in ESA that make this region unlikely to be membrane spanning.


Fig. 2. Flotillin is homologous to ESA and to two Synechococcus proteins. A, sequence alignment of flotillin with the mouse and human ESA proteins. Residues that are identical between flotillin and either mouse or human ESA are boxed. A/G, E, A/G, E repeat motifs are shaded. B, sequence alignment of flotillin with two hypothetical proteins from Synechococcus. Residues that are identical between flotillin and either ORF1 or ORF2 are boxed. Two conserved A/G, E, A/G, E repeat motifs are shaded.
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Homology searches also reveal a surprising similarity between flotillin and two conceptual proteins from the cyanobacterium Synechococcus PCC7942 (Fig. 2B). The homology extends for nearly the entire lengths of flotillin and the ORF2 protein. Two A/G, E, A/G, E motifs are partially conserved between flotillin and the Synechococcus ORFs (shaded boxes). 24% of the residues in ORF2 protein are identical to their corresponding residues in flotillin. In the region of highest homology (residues 84-240 of flotillin) the amino acid sequence of flotillin is 30% identical to that of ORF2 and 58% identical or conservatively substituted. However, no such homologies are detected between flotillin and the 6-frame translation of the completely sequenced yeast genome.

Tissue-specific Expression of Flotillin Messenger RNA

Because flotillin was initially identified by its co-purification with caveolin-1, flotillin mRNA should be expressed in tissues that also express caveolins. Northern blot analysis reveals that flotillin is expressed at the mRNA level in white adipose tissue, heart muscle, skeletal muscle (diaphragm), and lung (Fig. 3). As predicted, these results are similar to the expression pattern of caveolin-1 (37); however, flotillin is expressed at higher levels in heart relative to lung than is caveolin-1. Longer exposures of the autoradiogram also show low levels of expression in spleen, liver, and testis (data not shown). Of particular note is that flotillin mRNA is easily detectable in brain. In contrast, there is no detectable mRNA for any known caveolin family member expressed in brain (37-39). ESA mRNA has been detected in tissues that contain epithelia, such as pancreas, stomach, and lung, in cultured keratinocytes and melanocytes, and at low levels in heart (20).


Fig. 3. Northern blot analysis of the tissue-specific expression of flotillin. Poly(A)+ RNA (1 µg) isolated from each tissue or cultured cell line was subjected to formaldehyde-agarose gel electrophoresis and transferred to nylon membrane. This membrane was hybridized with a radiolabeled probe corresponding to the flotillin coding sequence. The same membrane was probed for HSP70 to indicate relative loading of RNA. The lane labeled 3T3-L1 diff contains 0.5 µg of poly(A)+ RNA isolated from 3T3-L1 adipocytes at Day 8 of differentiation.
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Characterization of the Flotillin and ESA Proteins

To characterize flotillin at the protein level, we generated an anti-peptide polyclonal antiserum in rabbits. The epitope within flotillin was chosen based upon its predicted antigenicity, surface probability, and the specificity of its sequence. Affinity-purified immune antibodies recognize a 47-kDa protein in Western blots of caveolin-rich membrane domains isolated from human lung (data not shown) and from 3T3-L1 adipocytes (Fig. 4). Preimmune sera do not recognize this band. Furthermore, the immune antibodies do not recognize the 47-kDa protein in the presence of the peptide that was used for immunization but do recognize this protein in the presence of irrelevant peptide. We have not yet found conditions under which our polyclonal flotillin antibody is able either to immunoprecipitate flotillin or to demonstrate specific, reproducible staining by immunofluorescence.


Fig. 4. Characterization of affinity-purified polyclonal antisera raised against a flotillin peptide. Caveolin-rich membrane domains were isolated on the basis of their low density and Triton X-100 insolubility as described under "Experimental Procedures." These domains were boiled in Laemmli sample buffer under reducing conditions, resolved by preparative SDS-PAGE, and transferred to nitrocellulose. Strips of this preparative membrane were incubated with preimmune sera (P) or with immune sera (I), in the absence of competing peptide (0), in the presence of a 100-fold molar excess of the peptide against which the immune sera were raised (K), or in the presence of a 100-fold molar excess of an irrelevant peptide (F). Detection was accomplished with an enzyme-linked secondary antibody and enhanced chemiluminescence.
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Based upon the two potential transmembrane domains in the predicted primary amino acid sequence (Fig. 1), we predicted that flotillin would associate tightly with membranes. Peripheral membrane proteins dissociate from membranes at high pH, but integral membrane proteins remain attached (28). When 3T3-L1 adipocytes were lysed at high pH in carbonate buffer and then subjected to centrifugation, immunoreactive flotillin was present in the membrane pellet, as was another integral membrane protein, caveolin-1 (Fig. 5). GDI-1 (a guanine-nucleotide dissociation inhibitor for Rab family proteins) is a soluble marker and was present predominantly in the supernatant. Interestingly, despite the absence of a predicted transmembrane domain in ESA, immunoreactive ESA was detected exclusively in the pellet with flotillin and caveolin-1. These data collectively indicate that flotillin and ESA behave as integral membrane proteins.


Fig. 5. Flotillin and ESA are integral membrane proteins. 3T3-L1 adipocytes were subjected to alkaline extraction as described under "Experimental Procedures" and analyzed by Western blot with 1) anti-flotillin antibodies, 2) anti-ESA monoclonal antibody, 3) anti-GDI-1 sera, and 4) anti-caveolin-1 monoclonal antibody. Guanine nucleotide dissociation inhibitor (GDI-1) is a marker for soluble proteins. Caveolin-1 is a marker for integral membrane proteins; S, supernatant (soluble proteins and peripheral membrane proteins); P, pellet (integral membrane proteins).
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We have shown previously that caveolin-1 mRNA and protein levels, as well as the number of caveolae per cell, increase during the differentiation of 3T3-L1 fibroblasts into adipocytes (37). At the mRNA level, induction of both caveolin-1 and caveolin-2 occurs between Day 2 and Day 4 of differentiation (37, 38). In contrast, the pattern of induction of flotillin mRNA is strikingly different (Fig. 6A). By Day 0, at which time the 3T3-L1 cells have been confluent for 2 days, flotillin mRNA expression already has increased to near-maximal levels. Flotillin mRNA levels then fall to a nadir by Day 2, only to increase again to maximal levels by Day 4. RNA levels for flotillin then remain relatively constant through Day 8. We have noted a similar decrease in mRNA levels for beta 2-microglobulin on Day 2 of 3T3-L1 adipocyte differentiation,4 which may reflect a transcriptional response to one or more components of the media used for differentiation from Day 0 to Day 2. 


Fig. 6. Flotillin expression during 3T3-L1 adipocyte differentiation. A, Northern blot: each lane contains 0.5 µg of poly(A)+ RNA isolated from 3T3-L1 cells at sequential stages of differentiation into adipocytes. The Northern blot was prepared and hybridized with a flotillin probe and HSP70 probe as described in the legend for Fig. 3. B, Western blot: 50 µg of proteins extracted from 3T3-L1 cells at sequential stages of differentiation were resolved by SDS-PAGE and transferred to nitrocellulose. Equal loading of protein was documented by Ponceau S staining (top panel). The membrane was blotted with antibodies to flotillin (2nd and 3rd panels from the top), ESA, and Acrp30. Flotillin (filled arrowhead) migrates as a 47-kDa protein and is induced at least 10-fold during adipogenesis. Anti-flotillin antibodies also recognize a ~45-kDa protein (unfilled arrowhead) and a 27-kDa protein (FCRD). Acrp30 is a specific marker for adipocyte differentiation that, like other such marker proteins (Glut4), first appears on Day 4 of 3T3-L1 differentiation (27).
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At the protein level, flotillin more closely follows the induction pattern of caveolin-1 (filled arrowhead, Fig. 6B). Between Day 2 and Day 4 flotillin protein levels per mg of total protein in post-nuclear cell lysates increase by ~3-fold. There is a further ~2-fold increase from Day 4 to Day 6 and another 2-fold increase from Day 6 to Day 10. A parallel induction pattern is observed for a ~27-kDa immunoreactive protein. We do not know whether this is a cross-reacting protein, a degradation fragment of flotillin, or a physiologically cleaved fragment of flotillin. Hence, we have termed this ~27-kDa protein FCRD (flotillin cross-reacting determinant). There is another protein of ~45 kDa (open arrowhead, Fig. 6B) that is recognized by the polyclonal flotillin antibody on a Western blot of 3T3-L1 adipocytes during differentiation. In contrast to flotillin and FCRD this ~45-kDa protein reaches peak levels on Day 4. Because of its different pattern of expression during 3T3-L1 adipocyte differentiation, the ~45-kDa protein is likely a cross-reacting protein that is recognized by the anti-flotillin polyclonal antiserum but is unrelated to either flotillin or ESA.

Whereas flotillin protein is induced during 3T3-L1 adipocyte differentiation, ESA protein remains at constant levels per mg of protein in post-nuclear cell lysates (Fig. 6B).

Flotillin Is an Integral Membrane Component of Caveolae Membranes

To evaluate the extent of specific localization of flotillin and ESA in caveolae, several complementary methods were used to purify caveolae membranes from 3T3-L1 adipocytes and endothelial cells. In all three methods, immunoblotting with anti-caveolin-1 IgG was used to track the position of caveolae-derived membrane domains.

First, proteins from 3T3-L1 adipocytes were fractionated using an established protocol that separates caveolin-1 and other caveolae-associated proteins from the bulk of cellular proteins and membranes. This procedure is based upon (i) the insolubility of caveolae membranes in the detergent Triton X-100 at 4 °C and (ii) their specific buoyant density in equilibrium sucrose gradients. With this fractionation method, we have previously shown that endogenous caveolin-1 (fractions 5-6) is purified ~2000-fold relative to total cell lysates. In addition, caveolin-1 is separated from greater than 99.95% total cellular proteins and compartment-specific markers for endoplasmic reticulum, Golgi, lysosomes, mitochondria, and non-caveolar plasma membrane, which remain in fractions 9-13 of these bottom-loaded sucrose density gradients (19, 30, 31).

Fractions from these gradients were harvested from the top, and the proteins of each fraction were resolved by SDS-PAGE, transferred to nitrocellulose, and blotted with the flotillin antibody and monoclonal antibodies against ESA and caveolin-1. The top panel of Fig. 7A shows the distribution of total cellular protein across the gradient by Ponceau S staining. Immunoreactive flotillin, ESA, and caveolin-1 co-fractionate and are specifically concentrated within fraction 5, which corresponds to caveolin-rich membrane domains that can be visualized as a light-scattering band in the gradient (Fig. 7, A and B). Significantly, flotillin, ESA, and caveolin-1 are clearly separated from the bulk of cellular proteins, which remain within fractions 9-12 and the pellet. FCRD remains in fractions 10, 11, and 12 and does not co-fractionate with caveolin-1.


Fig. 7. Flotillin and ESA co-fractionate with caveolin-1 in adipocytes, and flotillin is a marker for the Triton-insoluble, buoyant membrane fractions in brain tissue. A and B, 3T3-L1 adipocytes were homogenized in the presence of Triton X-100 and fractionated by relative buoyancy in sucrose gradients as described under "Experimental Procedures." The fractions were harvested from the top and analyzed by SDS-PAGE and Western blotting. Proteins were detected by Ponceau S staining. Specific proteins were detected with anti-flotillin and anti-caveolin-1 antibodies (A) and, in an independent experiment, with anti-ESA and anti-caveolin-1 antibodies (B). C, 3T3-L1 adipocytes were homogenized in the absence of detergent in carbonate buffer and fractionated by relative buoyancy in a sucrose gradient as described under "Experimental Procedures." Fractions were then harvested and analyzed as described above for A and B. D, bovine brain tissue (cortical gray matter harvested by suction) was homogenized in the presence of Triton X-100 and further processed as described for A and B.
[View Larger Version of this Image (33K GIF file)]

Next, we also used a detergent-free protocol to purify caveolin-rich membrane domains as we have reported previously (14). This protocol substitutes the nonionic detergent Triton X-100 with sodium carbonate to disrupt cellular membranes. Using this method, caveolin is highly enriched in a buoyant, light-scattering band just as with the Triton-based method, but in contrast to the Triton-based method, the glycosylphosphatidylinositol (GPI)-anchored protein carbonic anhydrase IV is detected in less buoyant fractions. We applied this carbonate-based method to the fractionation of 3T3-L1 adipocytes, and flotillin, ESA, and caveolin-1 again co-purified (Fig. 7C) in the buoyant, light-scattering fraction that corresponds to caveolae-enriched membranes.

Finally, we used a third independent method for the purification of caveolae in a different cell type to confirm the membrane compartment in which flotillin and ESA reside. In this method, the rat lung vascular system is perfused with cationic colloidal silica particles (32). These particles adhere to the luminal plasma membranes of endothelial cells and increase their density such that they can be highly purified by homogenization and centrifugation. The resulting membrane pellet retains caveolae on the side of the membrane opposite the silica. These caveolae are then sheared off of the plasma membrane-silica complex (P) by homogenization at 4 °C in the presence of the detergent Triton X-100. The caveolae (V) are then purified away from the stripped plasma membrane-silica complex (P - V) by sucrose gradient centrifugation. With this method >95% of the caveolin signal is present in caveolae, whereas >95% of the signals for the GPI-anchored proteins 5'-nucleotidase and urokinase-plasminogen activator receptor are present in P - V. Thus, this method can be used to distinguish between two Triton-insoluble membrane domains as follows: (i) caveolae, which are enriched in the marker protein caveolin, and (ii) plasma membrane domains that are enriched in GPI-anchored proteins. Western blot analysis of these three fractions (P, V, and P - V) demonstrated that flotillin, ESA, and caveolin-1 are highly enriched in V, the purified caveolae (Fig. 8, A and B). Only at nonlinear exposures of V was any signal for flotillin also detectable in P - V (middle panel of Fig. 8A). Notably, FCRD is detected only in the P - V fraction and is excluded from the V fraction.


Fig. 8. Plasma membrane flotillin and ESA in lung endothelial cells reside in the caveolae vesicles. Endothelial cell luminal surface plasma membranes from rat lung were isolated using cationic silica particles as described (32). This plasma membrane fraction (P) was further fractionated into caveolae (V) and plasma membranes stripped of caveolae (P - V). Equal amounts of protein (5 µg) from each fraction (P, V, and P - V) were analyzed by SDS-PAGE, Western blotting, and enhanced chemiluminescence. A, top panel: with a linear exposure, flotillin is only detected in the caveolae fraction (V). A, middle panel: at nonlinear exposures for flotillin in V, flotillin can also be detected in the P and P - V fractions, as can FCRD. A, bottom panel: the same Western blot decorated with an anti-caveolin-1 antibody shows a trace of caveolin-1 in the P - V fraction only at prolonged exposures. B, in an independent experiment, plasma membrane ESA and caveolin-1 co-localize almost exclusively within the caveolar fraction (V).
[View Larger Version of this Image (31K GIF file)]

Taken together, these data indicate that flotillin and its homologue ESA are resident integral membrane protein components of caveolae.

Flotillin Is a Marker for the Buoyant, Triton-Insoluble Membrane Fraction in Brain

As noted above, flotillin mRNA is easily detectable in mouse brain by Northern blot analysis (Fig. 3), whereas mRNA species for known caveolin family members are undetectable. This observation led to the question of whether flotillin is expressed at the protein level in brain and, if so, whether it sediments in the buoyant, Triton-insoluble membrane fractions. To address these issues, bovine brain tissue (cortical gray matter) was subjected to the same Triton-based purification protocol that we used for 3T3-L1 adipocytes. Indeed, just as is true in adipocytes, flotillin is present in the buoyant, Triton-insoluble fractions in bovine brain tissue (Fig. 7D). Current data do not specify in what cell type or types flotillin is expressed in bovine brain. However, flotillin is detectable in the buoyant, Triton-insoluble membrane fractions of PC12 cells (data not shown), which are derived from a rat pheochromocytoma (a neuroendocrine tumor). This latter finding is suggestive, although not conclusive, that neurons express the flotillin protein.


DISCUSSION

In this report we have described a new gene family of caveolae-associated integral membrane proteins that so far includes flotillin and ESA. Multiple lines of evidence support the conclusion that flotillin and ESA are resident components of caveolae. First, peptides that are contained within the predicted amino acid sequences of flotillin and ESA have been identified by microsequence analysis of caveolin-rich membrane domains purified from mouse lung tissue. Second, an affinity-purified, anti-peptide antibody raised against an epitope of flotillin recognizes a 47-kDa protein that co-purifies with caveolin-1 in 3T3-L1 adipocytes using either detergent-based or detergent-free purification protocols. Likewise, ESA co-purifies with caveolin-1 in the same assays. Third, caveolae vesicles that have been purified from rat lung endothelial cell plasma membranes following in situ coating with colloidal silica contain almost all of the plasma membrane flotillin and ESA proteins.

Caveolin and flotillin have similar tissue distributions at the mRNA level. However, one important exception to this observation is brain, where flotillin is easily detectable but caveolin family members are absent. We have demonstrated that flotillin protein is expressed in brain and that it fractionates in the Triton-insoluble, buoyant membrane fractions. Several recent reports suggest that caveolae or caveolae-like structures are present in neural tissues (8, 9, 40). Thus, flotillin may represent a new marker for these structures in brain. Whether flotillin-rich membrane domains in neural tissues are functionally related to caveolin-rich membrane domains in adipocytes and other caveolin-expressing tissues will be addressed in future experiments.

The anti-peptide polyclonal antisera we have raised against flotillin also recognizes a polypeptide of approximately 27 kDa that we have termed FCRD. Current data do not permit elucidation of the exact origins of FCRD. One possibility is that FCRD is a degradation product of flotillin that is produced during sample preparation. Another possibility is that it is a physiologically relevant proteolytic fragment of flotillin. Finally, FCRD may be an intact flotillin homologue or a completely unrelated protein that simply shares a conserved epitope. However, our current data highlight several important features of FCRD. First, FCRD is present in the plasma membrane fractions of rat lung endothelial cells (P and P - V), but it is absent from the caveolar fraction (V). Second, FCRD is soluble in Triton X-100 in adipocytes and, accordingly, does not co-purify with caveolin-1. Finally, FCRD polypeptide levels increase during 3T3-L1 adipocyte differentiation in parallel to those of flotillin. These data are all consistent with a model in which caveolar flotillin is proteolytically cleaved into FCRD and other fragment(s), with the result that FCRD leaves the caveola. If this model is correct, then the full description of FCRD at the amino acid level may lead to the identification of motifs important for caveolar localization and retention.

The close homology between flotillin and ESA and their co-purification with caveolin-1 in 3T3-L1 adipocytes suggest that flotillin and ESA are closely related members of the same gene family. In light of these findings ESA now seems to be an inappropriate appellation because ESA is expressed in cells as divergent as keratinocytes, adipocytes, and endothelial cells. To emphasize that ESA and flotillin constitute a newly recognized gene family, we propose that flotillin henceforth be known as flotillin-1 and ESA be known as flotillin-2. Whether additional members of the flotillin gene family exist is unknown.

A surprising result of these investigations is the identification of two hypothetical proteins of the cyanobacterium Synechococcus that share remarkable homology to flotillin. These homologies point to a very ancient and fundamental function for the flotillin gene family. Although Triton X-100 insoluble, buoyant membrane domains have been described in the yeast Saccharomyces cerevisiae (41), no convincing flotillin homologues have been identified in the S. cerevisiae Genome Data Base.

An important difference between flotillin and ESA is their patterns of protein expression during 3T3-L1 adipocyte differentiation. Whereas the ESA protein is expressed at constant levels as 3T3-L1 cells differentiate into adipocytes, the flotillin protein is induced at least 10-fold. Caveolin-1 mRNA and protein levels and caveolin-2 mRNA levels, as well as morphologically distinct caveolae, also increase significantly during adipogenesis (37, 38). The invariant levels of ESA protein during 3T3-L1 adipocyte differentiation suggest two possibilities as follows: (i) ESA may reside in a non-caveolar pool in preadipocytes and be recruited to caveolae during differentiation, or (ii) the concentration of ESA per caveola may decrease during differentiation.

Of note is the discordance between flotillin mRNA levels and protein levels during 3T3-L1 adipocyte differentiation as detected by Northern and Western blotting, respectively. Flotillin mRNA levels are roughly equal on days 0 and 8 of differentiation, but flotillin protein levels increase by an order of magnitude over the same period. Some other process during 3T3-L1 adipocyte differentiation has the effect of increasing the efficiency of flotillin mRNA translation or the stability of the translated flotillin protein or both. One attractive model is that flotillin protein is stabilized by the increased expression of caveolin protein during 3T3-L1 adipocyte differentiation, and/or by the assembly of caveolae, the number of which also increase during this differentiation program.

What is the function or functions of the flotillin gene family? Their localization to caveolae suggests some possibilities. First, like caveolin, flotillin and ESA may contribute to the structure of caveolae. One model for caveolin function holds that caveolin monomers assemble into homo-oligomers that pack side-by-side to form a scaffold onto which signaling molecules can attach (14, 17). The caveolin scaffold would then provide a mechanism for organizing these signaling molecules and sequestering them in a given functional state. Flotillin and ESA may be structural components of this scaffold or may form the backbone of a distinct part of the caveolae architecture, e.g. the caveolar neck. Another possible function for flotillin and ESA is that they participate in signaling, either as receptors or as membrane-bound effectors. Flotillin and ESA do share a conserved tyrosine phosphorylation site, but whether they are indeed phosphorylated at this site remains to be determined experimentally. Further investigations into the membrane topology of these proteins will be important to determine whether they are a potentially novel class of receptors. ESA, in particular, was cloned on the basis of its being a keratinocyte cell surface antigen. Thus far, the only proposed function for ESA is a role in cell adhesion. This hypothesis is based upon the ability of the ECS-1 monoclonal antibody to cause cell detachment when it is added to intact cultured keratinocytes. That flotillin also plays a role in cell adhesion is an intriguing possibility. Other proteins known to mediate or modulate cell-matrix interactions have been shown to localize in or near caveolae (urokinase-plasminogen-activator receptor) (42) or to interact with caveolin directly (beta 1 integrins) (43). Thus, there is precedence for the involvement of caveolae in cell adhesion.

Whatever the functions of flotillin and ESA, it will be interesting to determine why two such similar proteins are present in the same organelle. One possibility is that flotillin and ESA are functionally redundant. Alternatively, flotillin and ESA might perform similar functions in different subcellular locations. They could define, for instance, two distinct subpopulations of caveolae. Finally, flotillin and ESA may cooperate to execute a coordinated function.

In conclusion, (i) the molecular cloning of flotillin-1 and (ii) the identification of flotillin-1 and flotillin-2 (ESA) as components of caveolae provide important new avenues by which to explore the structure and function of caveolae organelles.


FOOTNOTES

*   This work was supported in part by National Institutes of Health Grant DK47618 (to H. F. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U90435[GenBank].


c   Supported by National Institutes of Health Physician Scientist Award DK02219.
d   Supported by a fellowship from the Swiss National Science Foundation. Current address: Dept. of Cell Biology, The Albert Einstein College of Medicine, 1300 Morris Park Ave., The Bronx, NY 10461.
f   Supported by the Beth Israel Foundation, a Grant-in-Aid from the American Heart Association, and National Institutes of Health Grants HL43278 and HL52766.
g   Supported by a National Institutes of Health FIRST Award GM-50443, a grant from the Elsa U. Pardee Foundation, and a grant from the W. M. Keck Foundation to the Whitehead Fellows Program. Current address: Dept. of Molecular Pharmacology, The Albert Einstein College of Medicine, Yeshiva University, 1300 Morris Park Ave., The Bronx, NY 10461.
i   To whom correspondence should be addressed: The Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142-1479. Tel.: 617-258-5216; Fax: 617-258-6768; E-mail: lodish{at}wi.mit.edu.
1   The abbreviations used are: GPI, glycosylphosphatidylinositol; ESA, epidermal surface antigen; Mes, 4-morpholineethanesulfonic acid; PBS, phosphate-buffered saline; FCRD, flotillin cross-reacting determinant; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; ORF, open reading frame; P, plasma membrane; V, caveolae.
2   As we report, "flotillin" is a specific marker for the set of proteins that float like a flotilla of ships in the Triton-insoluble, buoyant fraction (flotilla: Spanish, diminutive of flota, fleet; from Old French, flote; from Old Norse, floti, raft, fleet) from American Heritage Dictionary of the English Language (1975) American Heritage Publishing Co., Inc., New York, NY.
3   E. Wolf, P. S. Kim, and B. Berger, submitted for publication.
4   P. E. Bickel, P. E. Scherer, M. P. Lisanti, and H. F. Lodish, unpublished observations.

ACKNOWLEDGEMENTS

We thank Dr. Stefan Constantinescu and Ralph Lin for critical review of the manuscript, Dr. Andreas Stahl and Ethan Wolf for useful discussions, and Dr. Giulia Baldini for assistance in Northern blot analysis.


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