©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Caveolin Is Palmitoylated on Multiple Cysteine Residues
PALMITOYLATION IS NOT NECESSARY FOR LOCALIZATION OF CAVEOLIN TO CAVEOLAE (*)

(Received for publication, December 2, 1994; and in revised form, January 24, 1995)

Dennis J. Dietzen W. Randall Hastings Douglas M. Lublin (§)

From the Departments of Pathology and Internal Medicine, Division of Laboratory Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Caveolae are subdomains of the plasma membrane which concentrate cholesterol, glycosphingolipids, and glycosylphosphatidylinositol-linked proteins. It has recently been demonstrated that specific members of the Src family of protein tyrosine kinases require palmitoylation of NH(2)-terminal cysteine residues to localize in caveolae. Here we report that caveolin, an integral membrane protein which forms part of the coat of caveolae, also incorporates palmitate through linkage to cysteine residues. Caveolin contains only three cysteine residues which are all located on the COOH-terminal side of the hydrophobic transmembrane region. Immunofluorescent staining of cells transfected with caveolin indicated that, like the NH(2) terminus, this COOH-terminal region is located on the cytoplasmic side of the plasma membrane. Studies of cysteine substitution mutants showed that all three cysteines are capable of incorporating palmitate and that the juxtamembrane Cys residue is the predominant site of palmitoylation. Simultaneous mutation of all three cysteine residues in caveolin resulted in the loss of ability to incorporate palmitate; however, this did not affect localization of the protein. Thus, palmitoylation of cysteine residues in nonmembrane spanning Src family protein tyrosine kinases has different consequences than in the transmembrane protein caveolin.


INTRODUCTION

The plasma membrane of many cell types contains non-clathrin-coated invaginations referred to as caveolae or plasmalemmal vesicles. These organelles are enriched in glycosylphosphatidylinositol (GPI)(^1)-linked proteins, cholesterol, and glycosphingolipids compared with the bulk-phase membrane(1, 2) . Despite their description 40 years ago by Yamada (3) and Palade(4, 5) , caveolae have only recently been implicated in a number of processes important for inter- and intracellular communication. Folate, for example, is thought to enter cells through GPI-linked receptors localized to caveolae through a process referred to as potocytosis(6) . Caveolae may also be important in regulating cellular calcium concentration, since they have been shown to contain an inositol 1,4,5-triphosphate-sensitive calcium channel (7) and an ATP-dependent calcium pump(8) .

Caveolae have been implicated in a signal transduction pathway that is engaged by cross-linking of GPI-linked proteins on T cells. A mode of signal transduction was suggested by association of GPI-linked membrane proteins with specific members of the Src family of protein tyrosine kinases, p56 and p59(9, 10, 11) . We have recently shown that this association can be explained by their common inclusion in Triton X-100-insoluble domains which contain caveolae (12) . This study showed that inclusion of the GPI-anchored protein decay accelerating factor (DAF) in caveolae was mediated by its lipid anchor while inclusion of p59 was mediated by dual acylation of an amino-terminal Met-Gly-Cys motif, Gly^2 being required for cotranslational myristoylation and Cys^3 being necessary for incorporation of palmitate. Thus, two different structural motifs have been defined which localize proteins to caveolae. It is not clear how other proteins, specifically those anchored to the membrane by hydrophobic transmembrane domains, are localized to this organelle.

Caveolin(13) , also referred to as VIP21(14) , is a 22-kDa integral membrane protein originally described as a tyrosine-phosphorylated substrate in Rous sarcoma virus-transformed fibroblasts(15) . Caveolin is a component of the striated coat of caveolae(16) . Its gene encodes a 178-amino acid protein with an unusually long stretch of 33-40 hydrophobic amino acids in a predicted beta-sheet conformation(13) . Western blotting and metabolic labeling of caveolin has revealed a low molecular weight doublet and various high molecular weight forms attributed to self-association(13, 17, 18) . Two distinct membrane topologies have been reported for caveolin. Based on the protein topology rules of Hartmann et al.(19) and the accessibility of caveolin to cell surface labeling, Kurzchalia et al.(14) and Lisanti et al.(20) concluded that the NH(2) terminus of caveolin was cytoplasmic, and the COOH terminus was extracellular. On the other hand, antibodies directed at separate NH(2)- and COOH-terminal domains of caveolin stained BHK cells only after permeabilization, indicating that both ends of the molecule are cytoplasmic(17) . This disparity has not been resolved.

The current study was undertaken to examine the molecular structure of caveolin and analyze its determinants for localization to caveolae. Our data show that cytoplasmically oriented cysteine residues in the COOH-terminal domain of caveolin are modified by covalent attachment of palmitate. Collective mutation of all three cysteines in caveolin to serines eliminated palmitoylation but did not affect targeting of caveolin to caveolae.


MATERIALS AND METHODS

Reagents and Cells

Madin-Darby canine kidney (MDCK) cells and human embryonic kidney 293 cells were maintained in alpha-minimum Eagle's medium and Iscove's medium, respectively, supplemented with 10% fetal bovine serum, 2 mM glutamine, 50 µg/ml streptomycin, and 50 units/ml penicillin in a 5% CO(2), 95% air atmosphere at 37 °C. The mouse monoclonal antibody to caveolin was obtained from Transduction Laboratories (Lexington, KY). Antibodies against the HA (12CA5) and c-Myc (9E10) epitopes were described previously(21, 22) . The rabbit polyclonal antibody to human DAF (DL6) was described in (23) . [9,10-^3H]Palmitic acid (56 Ci/mmol) and [9,10-^3H]myristic acid (16 Ci/mmol) were obtained from DuPont New England Nuclear.

DNA and Transfections

The canine caveolin cDNA (14) was obtained from Paul Dupree (European Molecular Biology Laboratory, Heidelberg, Germany). An epitope tagged version of caveolin was generated using polymerase chain reaction primers which encoded the HA epitope at the NH(2) terminus (YPYDVPDYAS) and the c-Myc epitope at the COOH terminus (EQKLISEEDL) and inserted into the expression vector pcDNA3 (Invitrogen, San Diego, CA). Individual cysteines (Cys, Cys, and Cys) were changed to serine residues through inverse polymerase chain reaction using oligonucleotide primers encoding the appropriate point mutation and wild-type plasmid as template. Double Cys Ser mutants were created by using the appropriate combinations of template DNA containing a single Cys Ser mutation and oligonucleotide primers containing the desired second mutation. Simultaneous mutation of all cysteine residues was accomplished by using doubly mutated plasmid DNA as a template to generate the triply mutated plasmid (Cys). All mutations were confirmed by DNA sequence analysis. 293 cells were transfected with the DNA of interest using lipofectamine (Life Technologies, Inc.) for 2 h at 37 °C. Transient transfectants were analyzed 48 h later. Stable transfectants were selected using neomycin resistance.

Biosynthetic Labeling and Immunoprecipitation

Plates containing 2-3 times 10^7 MDCK or 293 cells were washed with serum-free media and labeled with 0.5 mCi/ml [9,10-^3H]palmitic acid for 60 min at 37 °C. Cells were then lysed in 1 ml of buffer containing 60 mM octyl glucoside, 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM sodium orthovanadate, 5 mM EDTA, 10 mM iodoacetamide, and 10 µg/ml each of leupeptin and aprotinin. Lysates were centrifuged for 10 min at 12,000 times g, and the supernatants were precleared with nonspecific serum and the immunoabsorbent Pansorbin (Calbiochem). Caveolin was immunoprecipitated with a mouse monoclonal antibody and rabbit anti-mouse IgG followed by Pansorbin. Immunoprecipitates were washed three times in lysis buffer and eluted into Laemmli sample buffer and analyzed on sodium dodecyl sulfate-12% polyacrylamide gels (SDS-PAGE). In some experiments, replicate gel slices were soaked in either 1 M Tris (pH 7.5) or 1 M hydroxylamine (pH 7.5) for 16 h before fluorography. All fluorographs were generated by exposure for 10-14 days.

Western Blotting

Proteins were transferred to polyvinyl difluoride or nitrocellulose membranes and blocked for 60 min in Tris-buffered saline, 0.05% Tween 20, and 3% non-fat dry milk. After a 30-min incubation with primary antibody diluted in blocking buffer, the blot was incubated for 30 min in the presence of appropriate secondary antibody conjugated to horseradish peroxidase. Bands were visualized with ECL chemiluminescent substrate (Amersham Corp.).

Analysis of Thioester-linked Fatty Acids

Following biosynthetic labeling, immunoprecipitation, and separation by SDS-PAGE, proteins were transferred to polyvinyl difluoride membranes. The caveolin band was localized by Western blotting, excised, and subjected to hydrolysis in 1.5 N NaOH for 16 h at 37 °C. Following acidification to pH 1-2, toluene extracts of the hydrolysate were combined and dried under N(2). Material was re-dissolved in ethanol and analyzed by C-18 reversed phase thin layer chromatography with a mobile phase of glacial acetic acid:acetonitrile (1:1). Sections of the lane containing the caveolin hydrolysate were scraped and analyzed in a liquid scintillation counter. Migration of authentic [^3H]palmitate and [^3H]myristate was determined by autoradiography.

Immunofluoresence

Orientation of caveolin was determined by accessibility of antibodies to the HA and c-Myc epitopes at the NH(2) and COOH terminus of caveolin, respectively. Transfected 293 cells were plated on glass coverslips, fixed for 15 min in 4% paraformaldehyde, and treated with rabbit anti-DAF (DL6) and either mouse anti-HA or mouse anti-c-Myc antibodies in the presence or absence of 0.1% Triton X-100. Rhodamine-conjugated anti-mouse IgG and fluorescein-conjugated anti-rabbit IgG (Sigma) were then added before mounting with vectashield (Vector Laboratories, Burlingame, CA). Cells were examined and photographed at times 300 magnification using filters sensitive to each of the conjugated fluorophores.

Isolation of Caveolin-rich Membrane Fractions

Triton X-100-insoluble membrane fractions containing caveolae were isolated by Sepharose 4B chromatography as described by Cinek and Horejsi (24) and modified in(12) . Material eluting in fraction 2 (void volume) represents high molecular weight (>10^7 daltons) aggregates which contain markers of caveolae, whereas fractions 4 and 5 (included volume) contain detergent-soluble proteins.


RESULTS

Caveolin Is Modified by Thioester-linked Palmitate

Our recent finding that p56 and p59, two members of the Src protein tyrosine kinase family which reside in caveolae, are palmitoylated (12, 25) led us to consider the ability of caveolin to incorporate palmitate. To investigate this possibility, MDCK cells were incubated in the presence of [^3H]palmitate for 60 min and lysed in octyl glucoside. Following immunoprecipitation of caveolin and SDS-PAGE, two radiolabeled bands of approximately 24 and 25 kDa were detected by fluorography (Fig. 1). Incorporation of radiolabel into the two bands was proportional to the relative intensity of each band observed by Western blotting (not shown), indicating that neither form of caveolin was selectively labeled. The thioester nature of the fatty acid linkage was confirmed by treatment with neutral hydroxylamine. Amide-linked fatty acid is not sensitive to such treatment, whereas thioester bonds are. Hydroxylamine treatment removed most of the incorporated label from caveolin (Fig. 1). Because fatty acids may interconvert during metabolic labeling, we also cleaved incorporated fatty acid from caveolin by alkali treatment and examined the material by reversed phase TLC. The cleaved ^3H label from caveolin comigrated with authentic palmitate, confirming the identity of the fatty acid (Fig. 2). Thus, we conclude that caveolin is modified by thioester-linked palmitate.


Figure 1: Incorporation of [^3H]palmitate into caveolin and its sensitivity to neutral hydroxylamine. MDCK cells were incubated with [^3H]palmitate for one h and lysed as described under ``Materials and Methods.'' Duplicate samples of caveolin immunoprecipitates from the lysate were analyzed by SDS-PAGE and soaked overnight in either 1 M Tris (pH 7.5) or 1 M hydroxylamine (pH 7.5) followed by fluorography.




Figure 2: Identification of fatty acid removed from caveolin as palmitate. Following metabolic labeling with [^3H]palmitate, caveolin was immunoprecipitated from an MDCK lysate, separated by SDS-PAGE, transferred to polyvinyl difluoride membrane, and subjected to alkaline hydrolysis. Fatty acid extracted from the hydrolysate was analyzed by C-18 reversed phase TLC. The graph shows counts/min from the lane containing the caveolin hydrolysate at indicated distances from the origin. Migration of [^3H]myristate (C14:0) and [^3H]palmitate (C16:0) standards was determined by autoradiography and is indicated above the graph.



The COOH Terminus of Caveolin is Cytosolic-The likely targets for palmitoylation in caveolin are the three cysteine residues in the protein, all located on the COOH-terminal side of the putative transmembrane domain. Depending on the membrane orientation of caveolin, these cysteines may be extracellular or intracellular. To determine the orientation of this region, 293 cells were transfected with cDNA encoding caveolin with HA and c-Myc epitopes located at the extreme NH(2) and COOH terminus, respectively. Presence of the HA and c-Myc epitopes added approximately 5 kDa to the apparent molecular mass of caveolin as determined by SDS-PAGE and Western blot. The occurrence of a 30-kDa doublet was not the result of limited proteolysis as each band was recognized by antibodies toward both epitopes (Fig. 3). Both the epitope-tagged and wild-type version of caveolin localized to caveolae as determined by Sepharose 4B column chromatography (not shown). Orientation of the epitope-tagged caveolin was determined by immunofluorescent staining of transfected cells by antibodies to each epitope in unpermeabilized 293 cells or 293 cells permeabilized with 0.1% Triton X-100. As shown in Fig. 4, the extracellular DAF epitope was unaffected by the presence of Triton (A and B), whereas staining with anti-HA occurred in the presence (D) but not the absence (C) of Triton. Likewise, Fig. 5shows that staining with anti-Myc occurred only in the presence (D) and not the absence (C) of Triton with anti-DAF staining remaining unaffected (A and B). Analysis of the same transfected 293 cells with the anti-epitope antibodies by fluoresence-activated cell sorting, in which only externally oriented epitopes would be accessible to antibody, was also negative (data not shown). Together, these results confirm that caveolin exists with both the NH(2) and COOH termini located intracellularly.


Figure 3: Recognition of epitope-tagged caveolin by anti-caveolin, anti-HA, and Anti-c-Myc antibodies. 293 cells expressing the caveolin containing the HA and c-Myc epitopes at the NH(2) and COOH termini, respectively, were lysed and analyzed by SDS-PAGE followed by Western blotting. Replicate lanes were probed with anti-caveolin antibody (lane 1), anti-HA (lane 2), and anti-c-Myc (lane 3).




Figure 4: Detection of the NH(2)-terminal HA epitope of caveolin by immunofluorescence requires prior permeabilization of cells with Triton. 293 cells expressing epitope-tagged caveolin were stained with rabbit anti-DAF and murine anti-HA antibodies in the absence (A and C) or presence (B and D) of 0.1% Triton X-100. Fields A and B were photographed under excitation light and filter appropriate to detect anti-DAF staining, whereas fields C and D were photographed under conditions appropriate to detect anti-HA staining.




Figure 5: Detection of the COOH-terminal c-Myc epitope of caveolin by immunofluorescence requires prior permeabilization of cells with Triton. 293 cells expressing the epitope-tagged caveolin were stained with rabbit anti-DAF and murine anti-c-Myc antibodies in the absence (A and C) or presence (B and D) of 0.1% Triton X-100. Fields A and B were photographed under excitation light and filter appropriate to detect anti-DAF staining, whereas fields C and D were photographed under conditions appropriate to detect anti-c-Myc staining.



All Three Cysteines in Caveolin Are Modified by Palmitate

To determine which cysteine residue(s) were involved in the palmitoylation of caveolin, individual cysteine residues or a combination of residues were changed to serine. These mutated constructs were transfected into 293 cells which were metabolically labeled with palmitate 48 h later. Fig. 6A shows that mutation of Cys (lane 2), Cys (lane 4), or Cys (lane 6) by themselves were not sufficient to completely abolish palmitate incorporation, indicating that more than one of the cysteine residues was a target for palmitoylation. To further resolve the locus of palmitate incorporation, caveolin mutants containing different combinations of two cysteine residues mutated to serines were constructed. Each of these mutants was able to incorporate palmitate, but to a lesser degree than the wild-type protein (Fig. 6B). Taking into account the relative levels of expression and intensity of labeling, the Cys Ser (lane 2) and Cys Ser (lane 3) mutations had more drastic effects on palmitate incorporation than the Cys Ser (lane 4). As the latter mutant contains a cysteine only at position 133, this juxtamembrane residue appears to be the predominant site of palmitoylation. Furthermore, simultaneous mutation of all three cysteines in caveolin (Cys) was required to completely abolish palmitoylation (Fig. 6A, lane 7, and Fig. 6B, lane 5), suggesting that each cysteine residue is a target for modification by palmitate.


Figure 6: All three cysteine residues in caveolin are targets for palmitoylation. 293 cells expressing wild-type or various mutated forms of epitope-tagged caveolin were labeled for 1 h with [^3H]palmitate and lysed as described under ``Materials and Methods.'' A shows the effect of mutating individual cysteine residues on palmitate incorporation. Caveolin immunoprecipitates from each of three separate labeling experiments were analyzed by SDS-PAGE and fluorography (shown above). 5% of the respective cell lysates were subjected to SDS-PAGE and Western blotting with anti-HA antibody. The section of the Western blot corresponding to the proper molecular mass of epitope-tagged caveolin is shown below the fluorographs. B shows similar experiments measuring the degree of palmitate incorporation following simultaneous mutation of two specified cysteine residues.



Thioester-linked Palmitate Is Not Required for Localization of Caveolin to Caveolae-We previously demonstrated that the presence of Cys^3, which is required for palmitoylation of p59, was both necessary and sufficient within the context of the Src family of kinases to determine localization to caveolae(12) . Ultrastructural and biochemical evidence indicates that membrane fractions enriched in caveolae can be isolated as high molecular weight, Triton-insoluble aggregates(26, 27) . These Triton-insoluble complexes have been recovered with the void volume eluate of Sepharose 4B columns (24) and in a low density complex with glycosphingolipids following equilibrium sucrose density gradient centrifugation(1) . Caveolin expressed endogenously in MDCK cells elutes with the void volume fraction following Sepharose 4B column chromatography(12) . Thus, to determine if palmitoylation of caveolin was necessary for its targeting to caveolae, 293 cells expressing the wild-type protein and the nonpalmitoylated version of caveolin (Cys) were lysed in 1% Triton X-100 and subjected to Sepharose 4B column chromatography. Epitope-tagged caveolin eluted primarily in fractions 2 and 3 with very small amounts found in fractions 4 and 5, consistent with location in caveolae (Fig. 7). The unknown modification which induces the caveolin doublet did not affect localization of the molecule to caveolae as both bands were recovered in the void volume. Most importantly, the Cys version of caveolin eluted with a pattern that was indistinguishable from the wild type. Following equilibrium sucrose density gradient centrifugation, both the wild-type and Cys versions of caveolin were also isolated from a low density fraction which contains protein components of caveolae in complex with glycosphingolipids and cholesterol (data not shown). Thus, in contrast to p59, caveolin does not require modification with palmitate for partitioning into caveolae.


Figure 7: Palmitoylation of caveolin does not affect its localization to caveolae. 293 cells expressing the wild-type or Cys versions of epitope-tagged caveolin were lysed in Triton X-100 and subjected to Sepharose 4B column chromatography. Column fractions 1-7 were analyzed by Western blotting with anti-HA antibody. Only the section of the Western blot corresponding to the proper molecular mass of epitope-tagged caveolin is shown. V(0) indicates the elution of void volume material from the column in fraction 2 as measured with intact erythrocytes. Soluble, nonaggregated proteins of 20 and 70 kDa ran with a peak in fraction 5.




DISCUSSION

We report for the first time that caveolin incorporates palmitate through a thioester linkage. This type of modification is post-translational, reversible, and has been demonstrated for a number of proteins which localize to caveolae including G protein alpha subunits (28, 29, 30) , and members of the Src family of protein tyrosine kinases, p56 and p59(12, 25, 31, 32, 33) . The pattern of palmitoylation in caveolin is unique in that acylation occurs on three widely dispersed targets, Cys, Cys, and Cys. In contrast, G protein alpha subunits and Src family kinases are palmitoylated on either a single cysteine residue or a pair of closely spaced cysteine residues.

The protein sequence motifs and enzymes which putatively mediate protein S-acylation have not been defined. Camp and Hofmann (34) have purified a bovine brain thioesterase which depalmitoylates H-Ras. Molecular cloning of this thioesterase, however, revealed that the protein was primarily secreted (35) and, therefore, not likely to act on caveolin or p59 as these are palmitoylated intracellularly. Membrane proximity appears to be a common attribute of palmitoylated cysteine residues. Cys of caveolin lies at the COOH-terminal end of the hydrophobic transmembrane region and is followed by hydrophobic residues at three of the next six positions. An examination of 6 amino acids both upstream and downstream of Cys and Cys reveals that 50% of these residues are also hydrophobic giving these regions an affinity for the plasma membrane. Likewise, p59 requires the membrane-anchoring influence of myristate at Gly^2 for palmitoylation(12) . Quesnel and Silvius (36) have recently shown that acylation of membrane proximal cysteine residues occurs in a cell-free environment, suggesting that membrane affinity may not only be necessary, but also sufficient to effect protein S-acylation. On the other hand, the specificity of acyl chain incorporation and rapid isoproterenol-mediated turnover of Galpha(s)-bound palmitate (37, 38, 39) implies that palmitoylation is catalytically regulated. Further work is required to clarify the mechanism(s) of attachment of thioester-linked fatty acids.

Thioester-linked palmitate is a component of one of the two independent signals thus far defined for localization of proteins to caveolae. In the case of p59, we have shown that palmitoylation of Cys^3 in conjunction with myristoylation of Gly^2 is both necessary and sufficient for its localization to caveolae(12) . Palmitoylation of p59 modestly strengthens its membrane association, but it is not yet clear how palmitoylation specifically targets p59 to caveolae. Palmitoylation alone is not sufficient to localize proteins to caveolae as a chimeric, type I transmembrane protein consisting of the ectodomain of DAF and the transmembrane sequence of an HLA class I molecule and p59 as the cytoplasmic domain, incorporates palmitate but does not localize to caveolae. (^2)The second signal for caveolar localization is the GPI anchor which has also been implicated in apical sorting in polarized cells. Replacement of the GPI anchor of DAF with a membrane-spanning domain results in a protein which does not partition into caveolae. The coordinate influence of these two signaling motifs results in the common inclusion of GPI-linked proteins and Src protein tyrosine kinases in caveolae, forming a basis for signal transduction through GPI-linked proteins. Furthermore, these signals apply to ``single-leaflet'' proteins that do not fully traverse the plasma membrane, that is, GPI-linked proteins insert only in the exoplasmic leaflet and Src family protein tyrosine kinases insert into the cytoplasmic leaflet of the plasma membrane. When and where the GPI anchor and the dually acylated Met-Gly-Cys motif of p59 exert their influence is only poorly understood. DAF becomes part of a Triton insoluble complex consisting of glycosphingolipids in the Golgi apparatus on its way to the plasma membrane(1) . Src family protein tyrosine kinases, however, possess no signal sequence and are excluded from this vesicular pathway from the Golgi to the membrane. It is not clear at what point molecules like p59 are included in caveolae.

Signals which target transmembrane proteins to caveolae are not known. Post-translational modification by palmitate was an intriguing possibility considering the increasing abundance of palmitoylated proteins in caveolae and its role in targeting Src kinases. Data presented here clearly indicate that palmitoylation does not play the same role in the targeting of caveolin as in the localization of p59 to caveolae. The precise role played by covalent attachment of palmitate in the function of caveolin must wait a further definition of that function. Caveolin is located on the cytoplasmic face of caveolae where it may be important in communicating with the intracellular environment, possibly through interaction with the cytoskeleton(16) . Fischer rat thyroid cells do not express caveolin, missort GPI-linked proteins to the basolateral surface, and do not form clusters between GPI-linked proteins and glycosphingolipids, suggesting the possible involvement of caveolin in these processes(40, 41) . Dissection of the domains involved in the targeting and function of caveolin will define new molecular signals important in the biogenesis and function of caveolae.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM 41297 (to D. M. Lublin), American Cancer Society Grant BE-201 (to D. M. Lublin), and National Institutes of Health Training Grant HL 07038 (to D. J. Dietzen). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pathology, Washington University School of Medicine, 660 S. Euclid Ave., Box 8118, St. Louis, MO 63110. Tel.: 314-362-8849; Fax: 314-362-3016.

(^1)
The abbreviations used are: GPI, glycosylphosphatidylinositol; DAF, decay accelerating factor; MDCK, Madin-Darby canine kidney cells; PAGE, polyacrylamide gel electrophoresis.

(^2)
J. Kwong, D. J. Dietzen, and D. M. Lublin, unpublished observation.


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