2544 Research Article

IntroductionMultiple sorting signals that reside at the N-terminus of thecytoplasmic domain of the chicken kidney AE1-4 anionexchanger direct the basolateral sorting and Golgi recycling ofthis membrane transporter in polarized MDCK cells (Adair-Kirk et al., 1999; Adair-Kirk et al., 2003). Recent studies haveshown that one of these signals, which is located betweenamino acids 38 and 63 of AE1-4, is sufficient to directbasolateral sorting when fused to a cytoplasmic tailless murineIgG FcRII B2 receptor. However, this chimeric receptor, Fc38-63, primarily accumulates in the trans-Golgi-network (TGN)at steady state. This localization profile is dependent uponrecycling of Fc38-63 from the basolateral membrane to theTGN. Mutagenesis studies have shown that the TGN recyclingof Fc38-63 is dependent upon a tyrosine residue located in thetetrapeptide Y47VEL, which matches the sequence of a YXX�motif, where X is any amino acid and � is a hydrophobicresidue. This motif directs the clathrin-dependent endocytosisof membrane-spanning proteins (Collawn et al., 1990) throughits association with the AP2 clathrin adaptor complex (Aguilaret al., 2001; Boehm and Bonifacino, 2001).

Other TGN resident proteins, such as furin (Schafer et al.,1995) and TGN38 (Bos et al., 1993; Wong and Hong, 1993),also undergo recycling from the cell surface to the TGN andtheir internalization from the cell surface requires the tyrosine-based endocytic signals YKGL and YQRL, respectively. Theendocytic signals of TGN38 and furin each bind the mu-subunit of the clathrin AP2 adaptor complex (Owen and Evans,1998; Teuchert et al., 1999). Furthermore, the endocytosis of

TGN38 and furin is dependent upon the tyrosine and thehydrophobic residue in their YXX� motifs (Humphrey et al.,1993; Owen and Evans, 1998).

Some lipids (Sharma et al., 2003; Singh et al., 2003) andproteins (Le et al., 2002) that recycle from the plasmamembrane to early compartments of the secretory pathway areinternalized by non-clathrin-dependent endocytic pathways.Cargo internalized by clathrin-independent pathways oftentraverse caveolin 1-positive endosomes prior to their deliveryto early secretory pathway compartments (Pelkmans et al.,2001; Nichols, 2002; Sharma et al., 2004). However, theendocytosis of these clathrin-independent cargos from theplasma membrane can occur through vesicular carriers coatedwith caveolin 1 or through uncoated vesicular carriers(Kirkham et al., 2005; Damm et al., 2005; Cheng et al., 2006).

In this report we have further characterized the recyclingpathway of Fc38-63 in MDCK cells. Studies using smallinterfering RNA (siRNA) and dominant-negative mutantsshow that even though the endocytosis of Fc38-63 is dependentupon a YXX� motif, it is internalized from the plasmamembrane through a caveolin-dependent pathway. Moreover,co-precipitation studies indicate that caveolin 1 is associatedwith Fc38-63 and the AE1-4 anion exchanger in this kidneyepithelial cell type. Mutations in the cytoplasmic Y47VELtetrapeptide of these proteins disrupt their interaction withcaveolin 1. The fact that these mutations also inhibit Fc38-63and AE1-4 trafficking suggests a novel role for YXX� motifsin targeting membrane-spanning proteins to caveolin-dependent sorting pathways.

Previous studies showed that the sequence between aminoacids 38 and 63 of the chicken AE1-4 anion exchanger issufficient to direct basolateral sorting and recycling to theGolgi when fused to a cytoplasmic tailless FcRII B2receptor. Further characterization of the recycling pathwayhas indicated that the chimera Fc38-63 colocalizes withcaveolin 1 in the basolateral membrane of MDCK cells, andin early endosomes following its internalization from thecell surface. Studies using small interfering RNA (siRNA)and dominant-negative mutants revealed that Fc38-63endocytosis is primarily caveolin-dependent and clathrin-independent. The endocytosis of the chimera is alsodependent upon cholesterol and dynamin. Co-precipitationstudies indicated that caveolin 1 associates with Fc38-63.

Mutation of the tyrosine or leucine residues in thecytoplasmic sequence Y47VEL of Fc38-63 disrupts thisinteraction and inhibits the endocytosis of the chimera.Additional analyses revealed that AE1-4 also associateswith caveolin 1. Mutation of the leucine in the Y47VELsequence of AE1-4 disrupts this interaction, and blocks therecycling of this transporter from the basolateralmembrane to the Golgi. The Y47VEL tetrapeptide matchesthe sequence of a YXX� motif, and our results indicate anovel role for this motif in directing caveolin-dependentsorting.

Key words: Caveolin, Sorting signal, Endosomes, AE1 anionexchanger

Summary

A novel role for a YXX� motif in directing the caveolin-dependent sorting of membrane-spanning proteinsFrank C. Dorsey*, Thangavel Muthusamy, Michael A. Whitt and John V. Cox‡

Department of Molecular Sciences, University of Tennessee Health Science Center, 858 Madison Avenue, Memphis, TN 38163, USA*Present address: Department of Biochemistry, St Jude Children’s Research Hospital, Memphis, TN, USA‡Author for correspondence (e-mail: jcox@utmem.edu)

Accepted 21 May 2007Journal of Cell Science 120, 2544-2554 Published by The Company of Biologists 2007doi:10.1242/jcs.002493

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2545Caveolin-dependent sorting

ResultsEps15-independent endocytosis of Fc38-63 isdependent upon a YXX� motifPrevious studies using chimeras between the chicken AE1-4anion exchanger and a cytoplasmic tailless murine IgG FcRIIB2 receptor defined multiple sorting signals within thecytoplasmic domain of AE1-4 (Adair-Kirk et al., 1999; Adair-Kirk et al., 2003). One of these signals, which resides betweenamino acids 38 and 63 of AE1-4, was sufficient to direct thebasolateral sorting and recycling of the AE1/Fc receptorchimera Fc38-63 from the plasma membrane to the TGN.Mutagenesis studies demonstrated that the steady-statelocalization of this chimera was altered when the tyrosine orleucine residues located in the sequence Y47VEL50 werechanged to an alanine. This tetrapeptide matches the sequenceof a YXX� motif that functions as a signal for clathrin-dependent endocytosis (Collawn et al., 1990).

To assess whether the tyrosine and leucine residues in theY47VEL50 sequence of Fc38-63 were involved in directing theendocytosis of this chimera, MDCK cells expressing wild-typeFc38-63, Fc38-63L50A or Fc38-63Y47A were incubated withan antibody that recognizes an extracellular epitope on the Fcreceptor for 30 minutes at 4°C to prevent endocytosis and thenshifted to 37°C for 45 minutes. As shown previously (Adair-Kirk et al., 2003), wild-type Fc38-63 internalized from the cellsurface and accumulated in a perinuclear compartment ofMDCK cells (Fig. 1A-C). By contrast, both Fc38-63L50A (Fig.1D-F) and Fc38-63Y47A (Fig. 1G-I) were almost entirelyretained in the basolateral membrane of these epithelial cells,indicating a crucial role for these residues in directingendocytosis.

The fact that Fc38-63 internalization was dependent uponboth tyrosine 47 and leucine 50 of its YXX� motif suggestedthat its uptake occurred in a clathrin-dependent fashion. Toexamine this possibility, we performed internalization assaysin cells expressing an enhanced green fluorescent protein(EGFP) fusion of dominant-negative Eps15, Eps15�95-295,which blocks clathrin-dependent endocytosis (Benmerah et al.,1999). The effect of this mutant on the endocytosis of thehuman transferrin receptor, a clathrin-dependent cargo, wasassessed by incubating MDCK cells expressing the receptorwith transferrin conjugated to Alexa Fluor-594 for 30 minutesat 4°C and then shifting the cells to 37°C for 30 minutes. Thisanalysis revealed that the endocytosis of the transferrinreceptor was blocked in cells that were also expressingEps15�95-295 (Fig. 2A-C). Conversely, expression ofEps15�95-295 did not affect the internalization of Fc38-63(Fig. 2D-F). Quantification of this assay revealed thattransferrin receptor endocytosis was entirely or almost entirelyblocked in 29 out of 30 cells expressing high levels ofEps15�95-295, whereas the endocytosis of Fc38-63 wasunaffected in 30 out of 30 cells expressing high levels of thisdominant-negative mutant. These data indicated that Fc38-63internalization can occur through a pathway that is dependentupon its cytoplasmic YXX� motif, but independent of clathrin.

The sorting of Fc38-63 in MDCK cells is cholesterol- anddynamin-dependentOther investigators have shown that the caveolar-dependentendocytosis of lipids (Sharma et al., 2003; Singh et al., 2003)and proteins (Le et al., 2002) is dependent upon cellular

cholesterol. Flask-shaped caveolae are not detected in theplasma membrane of cells treated with methyl-�-cyclodextrin,which binds cholesterol and depletes it from the plasmamembrane (Hao et al., 2001). To assess the role of cholesterolin the trafficking of Fc38-63, MDCK cells expressing thechimera were incubated in the presence of 10 mM methyl-�-cyclodextrin for 1 hour at 37°C. This treatment resulted in adramatic redistribution of Fc38-63, which substantiallyoverlaps the distribution of furin, a TGN marker, in untreatedcells (Fig. 3A-C). Although a fraction of Fc38-63 continued tooverlap the distribution of furin following methyl-�-cyclodextrin treatment (Fig. 3D-F), the majority of thechimeras were redistributed to the basolateral membrane wherethey overlapped the distribution of caveolin 1, a caveolar-

Fig. 1. MDCK cells expressing Fc38-63 (A-C), Fc38-63L50A (D-F)or Fc38-63Y47A (G-I) were incubated with rat anti-Fc receptorantibodies for 30 minutes at 4°C and then shifted to 37°C for 45minutes. Cells were then fixed, permeabilized and incubated withanti-rat IgG conjugated to Alexa Fluor-594 (A,D,G) and AlexaFluor-488-phalloidin (B,E,H). The confocal images correspond to anxy-slice through the middle of the cells. Bars, 10 �m.

Fig. 2. MDCK cells expressing Fc38-63 or the human transferrinreceptor were transfected with EGFP-tagged Eps15�95-295 (B,E).The cells were incubated with Alexa Fluor-594-transferrin (A) oranti-Fc receptor antibodies (D) for 30 minutes at 4°C and then shiftedto 37°C for 30 minutes. Cells were then fixed andimmunofluorescent molecules were directly visualized (A-C) or thecells were fixed, permeabilized and incubated with anti-rat IgGconjugated to Alexa Fluor-594 prior to microscopic analysis (D-F).Bars, 10 �m.

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associated protein (Fig. 4D-F), or to furin-negative intracellularmembranes. Many of the chimera-containing intracellularmembranes were also stained with antibodies to caveolin 1(Fig. 4D-F). The accumulation of Fc38-63 in the basolateral

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membrane of cells treated with methyl-�-cyclodextrinsuggested that cholesterol depletion inhibited the endocytosisof the chimera. Furin, like Fc38-63, recycles from the plasmamembrane to the Golgi, and its internalization from the cellsurface is dependent upon a YXX� sorting signal (Schafer etal., 1995). However, furin continued to accumulate inintracellular membrane compartments following cholesteroldepletion (Fig. 3F), indicating that the concentration of methyl-�-cyclodextrin used in these studies is not sufficient to inhibitits endocytosis.

Cellular cholesterol was replenished in methyl-�-

Fig. 3. MDCK cells expressing Fc38-63 were incubated in serum-free DMEM that lacked (A-C) or contained (D-F) 10 mM methyl-�-cyclodextrin for 1 hour. Cells were then fixed, permeabilized andincubated with rat anti-Fc-receptor antibody and with a rabbitantibody directed against furin. After washing, cells were incubatedwith anti-rat IgG conjugated to Alexa Fluor-594 and anti-rabbit IgGconjugated to Alexa Fluor-488. Alternatively, after a 1-hourincubation in 10 mM methyl-�-cyclodextrin, the cells were washedwith DMEM containing 5% fetal bovine serum and incubated for 20minutes (G-I) or 60 minutes (J-L) with 10 mM methyl-�-cyclodextrin loaded with cholesterol and processed as describedabove. The upper panel in each confocal image corresponds to an xy-slice near the middle of the cells, whereas the lower panelcorresponds to an xz-slice. Black bars mark the position of the basalmembrane. Bars, 10 �m.

Fig. 4. MDCK cells expressing Fc38-63 were processed as describedin the legend to Fig. 3 except the cells were incubated with caveolin1 antibodies rather than antibodies to furin. The upper panel in eachconfocal image corresponds to an xy-slice near the middle of thecells, whereas the lower panel corresponds to an xz-slice. Black barsmark the position of the basal membrane. Bars, 10 �m.

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2547Caveolin-dependent sorting

cyclodextrin-treated cells by incubating the cells in thepresence of soluble cholesterol/methyl-�-cyclodextrincomplexes. Treatment of cholesterol-depleted MDCK cells inthis way stimulated the internalization of the basolateralpopulation of Fc38-63. Within 20 minutes of adding cholesterolto the media, the surface population of Fc38-63 and caveolin 1internalized, and these proteins were colocalized in endosomes(Fig. 4G-I). Additional studies revealed that the majority of thesurface chimeras internalized within 3 minutes of addingcholesterol to the media (data not shown). One hour after theaddition of cholesterol, some of the chimeras still overlappedthe distribution of caveolin 1 (Fig. 4J-L). However, asignificant fraction of Fc38-63 assumed a TGN localizationprofile where it again overlapped the distribution of furin (Fig.3J-L). These data indicated that under the conditions of thischolesterol depletion/repletion protocol, some of the steps inthe trafficking of Fc38-63 in MDCK cells were linked to thetrafficking of caveolin 1.

The caveolar-dependent endocytosis of glycosphingolipidsis dependent upon dynamin (Puri et al., 2001), which isnecessary for the pinching off of caveolae from the plasmamembrane. To determine whether Fc38-63 endocytosisexhibited a similar dynamin-dependence, MDCK cellsexpressing the chimera were transfected with the dominant-negative dynamin 2(K44A) mutant. The transfected cells wereincubated with the Fc receptor antibody for 30 minutes at 4°Cand then shifted to 37°C for 30 minutes. This analysis revealedthat the internalization of Fc38-63 was almost entirely inhibitedin cells expressing dominant-negative dynamin (Fig. 5D-F),whereas similar studies with wild-type dynamin had no effecton chimera uptake (Fig. 5A-C). Taken together, our dataunexpectedly showed that Fc38-63 was internalized through apathway that was cholesterol- and dynamin-dependent andEps15-independent. These properties are hallmarks ofcaveolar-dependent endocytosis. The fact that Fc38-63internalization was also dependent upon its YXX� motifsuggested a novel role for this sorting signal in directing anendocytic pathway linked to caveolin 1.

Fc38-63 is endocytosed to caveolin 1-positiveendosomesThe Fc38-63 polypeptides that accumulated in the basalmembrane of methyl-�-cyclodextrin-treated cells significantlyoverlapped the distribution of caveolin 1 (Fig. 6A-C). Thisobservation was consistent with the hypothesis thatcholesterol depletion inhibited the caveolin-dependentendocytosis of the chimera. To investigate whether thecolocalization of Fc38-63 and caveolin 1 that was observedduring the cholesterol depletion/repletion protocol reflectedthe normal trafficking of these proteins, internalization assayswere performed. MDCK cells expressing the chimera wereincubated with the anti-Fc receptor antibody for 30 minutes at4°C to prevent endocytosis. At this time, the cells were eitherfixed (Fig. 6D-F) or shifted to 37°C for various times (Fig. 7)and the fate of the surface-labeled chimeras was followed byconfocal microscopy. This analysis revealed that the surfacepopulation of Fc38-63 overlapped the distribution of caveolin1 in the basolateral membrane of polarized MDCK cells (Fig.6D-F). Quantification of images similar to that shown in Fig.6F revealed that 65% of the surface pool of Fc38-63colocalized with caveolin 1 (0 minutes time point in Fig. 7D).

Consistent with our results obtained using the dominant-negative Eps15 construct (Fig. 2), quantitative colocalizationassays revealed that only 8% of surface Fc38-63 colocalizedwith clathrin (Fig. 7D). Additional studies indicated that 35%of surface Fc38-63 colocalized with the cholera toxin B (Ctx-B) subunit (Fig. 7D). Ctx-B binds the lipid GM1 ganglioside,which is expressed at detectable levels in a subset of MDCKcells, and is internalized from the cell surface through multiplepathways including caveolar-dependent endocytosis (Kirkhamet al., 2005).

Shifting the cells to 37°C for 2 minutes induced theendocytosis of the chimeras. The higher magnification imagesin Fig. 7A-C illustrate that a substantial fraction of theinternalized chimeras accumulate in caveolin 1-positiveendosomes. Quantification of this assay revealed that 61% of theinternalized chimeras accumulated in this caveolin 1-positiveendosomal compartment. Additional quantitative analysesrevealed that 33% of internalized Fc38-63 accumulated inendosomes containing Ctx-B that had also been endocytosedfrom the cell surface (Fig. 7D), whereas only 8% accumulatedin endosomes containing internalized transferrin (Fig. 7D). Thepercentage of Fc38-63 that accumulated in transferrin-positiveendosomes was identical to the percentage that colocalized withclathrin on the cell surface (Fig. 7D), suggesting that a smallpercentage of the chimeras are internalized by clathrin-dependent endocytosis and delivered to transferrin-positiveendosomes. Twenty percent of internalized Fc38-63 was presentin endosomes that were positive for EEA1 (Fig. 7D), a markerfor early endosomes that acquire cargo internalized by clathrinand caveolar-dependent endocytosis (Pelkmans et al., 2004). Thefact that internalized Fc38-63 minimally overlapped thedistribution of Ctx-B, transferrin and EEA1, which are markersfor both clathrin- and caveolar-dependent endocytosis, suggestedthe chimeras are endocytosed by a novel pathway. Five minutesafter the shift to 37°C, the percentage of internalized chimeraspresent in endosomes containing caveolin 1 and Ctx-B increasedslightly to 73% and 53%, respectively, whereas the chimeraspresent in endosomes containing transferrin and EEA1 wereessentially unchanged (Fig. 7D). The increased overlap between

Fig. 5. MDCK cells expressing Fc38-63 were transfected with wild-type dynamin 2 (A-C) or the dynamin 2 (K44A) mutant (D-F) in avector that also expresses EGFP. In each instance, the cells wereincubated with anti-Fc receptor antibodies for 30 minutes at 4°C andthen shifted to 37°C for 30 minutes. Cells were then fixed,permeabilized and incubated with anti-rat IgG conjugated to AlexaFluor-594. Bars, 10 �m.

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Fc38-63 and Ctx-B at the 5-minute time point suggests that thesecargo transit through a common caveolin 1-positive endosomalcompartment prior to their delivery to early compartments of thesecretory pathway (Adair-Kirk et al., 2003; Nichols, 2002).

Fc38-63 endocytosis is caveolin 1-dependentTo determine whether caveolin 1 is involved in the endocytosisof Fc38-63, an siRNA strategy was employed to downregulate

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caveolin 1 expression in MDCK cells. For these analyses, wetransiently expressed a caveolin 1 siRNA in MDCK cells usinga vector that expressed EGFP under the control of a separatepromoter. The sequence used for the siRNA has been used byother investigators to downregulate caveolin 1 expression in thiscell type (Manninen et al., 2005). Immunolocalization studiesrevealed that caveolin 1 expression levels were substantiallyreduced in cells expressing high levels of the EGFP reporter (Fig.8A-C). Quantification of images similar to this revealed that thecaveolin 1 siRNA reduced caveolin 1 expression levels to ~35%of that observed in non-transfected cells (Fig. 8G). The caveolinremaining in transfected cells probably represents a stablepopulation of caveolin that has been observed in previous studiesin which this same siRNA was used to downregulate caveolinexpression (Schuck et al., 2004). Additional studies using acontrol siRNA directed against EGFP had no effect on caveolin1 expression (data not shown).

To determine whether reducing caveolin 1 expressionlevels affected the endocytosis of Fc38-63, internalizationassays were performed in cells expressing the caveolin 1siRNA construct. These analyses revealed thatdownregulating caveolin 1 expression reduced Fc38-63endocytosis to ~18% of that observed in non-transfectedcontrol cells (Fig. 9A,B), whereas quantification of similarstudies revealed that the control EGFP siRNA had no effecton chimera internalization (Fig. 9B). The reducedendocytosis of Fc38-63 in cells expressing the caveolin 1siRNA was not because of a decrease in the surfaceexpression of the chimera, which increased slightly insiRNA-expressing cells (Fig. 8D-F,G).

Additional studies revealed that the caveolin 1 siRNA hadno effect on the endocytosis of transferrin (Fig. 9A,B), nor didit have any obvious effect on the endocytosis of Ctx-B (Fig.9A). However, the highly variable expression of the receptorfor the Ctx-B subunit, GM1 ganglioside, made it impossible toquantify the effect of the caveolin 1 siRNA on Ctx-B uptake.Nonetheless, these results demonstrate that the chimera isendocytosed by a caveolin-dependent pathway that is notnecessary for the endocytosis of Ctx-B.

Fig. 6. Polarized MDCK cells expressing Fc38-63 were incubated inthe presence of 10 mM methyl-�-cyclodextrin for 1 hour at 37°C.The cells were then fixed, permeabilized and incubated withantibodies specific for the Fc receptor (A) and caveolin 1 (B).Alternatively, untreated cells were incubated with anti-Fc receptorantibodies (D-F) for 30 minutes at 4°C. The cells were then washed,fixed, permeabilized and incubated with antibodies specific forcaveolin 1 (E). In both instances, the cells were washed andincubated with anti-rat IgG conjugated to Alexa Fluor-594 (A,D) andanti-rabbit IgG conjugated to Alexa Fluor-488 (B,E). Bars, 10 �m.

Fig. 7. Polarized MDCK cells expressing Fc38-63 were grownon permeable supports. The basolateral surface of the cellswas incubated with anti-Fc receptor antibodies (A) for 30minutes at 4°C. The cells were then washed and shifted to37°C for 2 minutes. After 2 minutes of incubation, the cellswere shifted to citrate buffer, pH 1.5, for 5 minutes at 4°C toelute surface-bound antibodies. The cells were then rinsed inPBS and fixed. Following permeabilization, the cells wereincubated with antibodies specific for caveolin 1 (B). The cellswere again washed and incubated with anti-rat IgG conjugatedto Alexa Fluor-594 (A) and anti-rabbit IgG conjugated toAlexa Fluor-488 (B). The merged image (C) illustrates theoverlap between the chimera and caveolin. The xy-slice ineach confocal image is approximately 1 �m above the basalmembrane of the cell. Similar analyses have quantified thecolocalization of Fc38-63 with clathrin, transferrin, Ctx-B orEEA1. The bar graph in D shows the percentage of the totalnumber of pixels resulting from Fc38-63 staining thatcolocalized with these various endocytic markers on the cellsurface (0 Min.), and at 2 and 5 minutes post-internalization.This quantification reflects the average result from at least 25cells from two independent experiments. Bar, 1 �m.

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2549Caveolin-dependent sorting

Association of Fc38-63 with caveolin 1 is dependentupon its YXX� motifThe data described above indicated that Fc38-63 endocytosis issignificantly impaired when caveolin 1 expression isdownregulated. To investigate whether an interaction betweenFc38-63 and caveolin 1 may be necessary for the endocytosisof the chimera, co-precipitation studies were performed.Because we were unable to immunoprecipitate Fc38-63 usingthe anti-Fc receptor antibody, we inserted a V5 epitope tag inthe extracellular domain of Fc38-63. Localization andinternalization assays with V5-tagged Fc38-63 revealed that theepitope tag did not alter the basolateral sorting of the chimera.Although the kinetics of endocytosis of V5-tagged Fc38-63

were slightly slower than the untagged chimera, V5-taggedFc38-63 traversed the same compartments as the untagged

Fig. 8. Subconfluent MDCK cells expressing Fc38-63 weretransfected with a vector expressing the caveolin 1 siRNA. Thisvector also expressed EGFP under the control of a separate promoter.Forty-eight hours post-transfection, the cells were fixed (A-C) orincubated with anti-Fc receptor antibodies for 30 minutes at 4°C,washed and then fixed (D-F). Following permeabilization, the cellsin A-C were incubated with caveolin 1 antibodies (A) followed byanti-rabbit IgG conjugated to Alexa Fluor-594. The cells in D-F weredirectly incubated with anti-rat IgG conjugated to Alexa Fluor-594.Following washing, cells were analyzed by confocal microscopy.Arrows in A and B mark the border of the cell expressing thecaveolin 1 siRNA. Note the absence of caveolin staining in theplasma membrane. The bar graph in G has quantified the averageeffect of the caveolin 1 siRNA on the caveolin 1 protein level andFc38-63 surface expression in 20 randomly chosen cells from twoindependent experiments. Bars, 10 �m.

Fig. 9. Subconfluent MDCK cells expressing Fc38-63 weretransfected with the caveolin 1 siRNA-expressing vector. Forty-eighthours post-transfection, the cells were incubated with anti-Fcreceptor antibodies, Ctx-B conjugated to Alexa Fluor-594 ortransferrin conjugated to Alexa Fluor-594 for 30 minutes at 4°C. Thecells were then washed and shifted to 37°C for 2 minutes. At thistime, the cells were shifted to citrate buffer, pH 1.5, for 5 minutes at4°C to elute surface-bound molecules. The cells were then rinsed inPBS and fixed. Following permeabilization, cells that had beenincubated with anti-Fc receptor antibodies were incubated with anti-rat IgG conjugated to Alexa Fluor-594. Immunofluorescentmolecules were visualized on a Zeiss LSM 510 confocal microscope.The bar graph in B has quantified the effect of the caveolin 1 siRNAon the endocytosis of Fc38-63 and transferrin in 20 randomly chosencells. The effect of a control siRNA directed against EGFP on Fc38-63 endocytosis is also illustrated (B). The standard error in B isindicated. Bars, 10 �m.

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chimera during recycling to the Golgi (data not shown).Immunoblotting analysis of a V5 immunoprecipitate preparedfrom cells expressing V5-tagged Fc38-63 revealed that thechimera migrated as a diffuse species ranging in size from ~47kDa to ~50 kDa (Fig. 10A). Similar blotting analysis of V5-

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tagged Fc38-63Y47A and Fc38-63L50A indicated that theseamino acid substitutions, which inhibited the endocytosis ofthe chimera, also altered the mobility of the proteins on sodiumdodecyl sulfate (SDS) gels. Fc38-63Y47A migrated as twospecies of ~42 kDa and ~45 kDa, whereas Fc38-63L50A

primarily migrated as two species of ~45kDa and ~47 kDa (Fig. 10A). To addressthe possibility that the protein profilesobserved in this blotting analysis reflectedheterogeneity in the acquisition of sugarson the four potential N-linkedglycosylation sites of the chimeras, the V5immunoprecipitates were digested withglycosidases to remove all sugars prior toblotting analysis. This experimentrevealed that each chimera migrated astwo species of ~32 kDa and ~35 kDafollowing glycosidase digestion (Fig.10A). These species were slightly largerthan the predicted molecular mass of theseproteins, which was ~29 kDa. Whether the~32 kDa or ~35 kDa species possessmodifications in addition to glycosylationis not known at this time. However, thesedata clearly indicate that the heterogeneityobserved for the wild-type and mutantchimeras was because of variability insugar acquisition. The reduced level ofsugar addition on Fc38-63Y47A and Fc38-63L50A relative to the wild-type chimeracorrelated with the reduced ability of themutants to undergo endocytosis andrecycling to the Golgi following surfacedelivery.

To determine whether Fc38-63associated with caveolin 1, V5immunoprecipitates prepared fromMDCK cells transfected with V5-taggedFc38-63 were probed with caveolin 1antibodies. This analysis revealed thatcaveolin 1 coprecipitated with V5-taggedFc38-63 (Fig. 10B). Similar studies usingcells transfected with V5-tagged Fc38-63Y47A and Fc38-63L50A indicated thatthese amino acid substitutions almostcompletely blocked the association ofcaveolin 1 with the chimera (Fig. 10B).Quantification of three independentexperiments revealed that the Y47Asubstitution resulted in a 99.2±1.4%reduction in the amount of caveolin 1 thatcoprecipitated with V5-tagged Fc38-63,whereas the L50A substitution resulted ina 97.9±3.6% reduction. The fact that thesemutant chimeras are also defective inendocytosis suggested that the YXX�-dependent interaction of the chimera withcaveolin 1 is necessary for its endocytosisfrom the plasma membrane. To ensure thatthe surface population of Fc38-63 doesindeed associate with caveolin 1, MDCK

Fig. 10. MDCK cells expressing V5-tagged versions of Fc38-63, Fc38-63Y47A, Fc38-63L50A were lysed and immunoprecipitates were prepared with V5-specific antibodies.The immunoprecipitates were incubated in the absence or presence of a mixture ofglycosidases that removes all O-linked and N-linked sugars prior to immunoblottinganalysis with V5 antibodies (A). V5 immunoprecipitates prepared from MDCK cellsexpressing V5-tagged versions of Fc38-63, Fc38-63Y47A, Fc38-63L50A were alsosubjected to immunoblotting analysis with V5- and caveolin 1-specific antibodies (B).Alternatively, cells expressing V5-tagged (+Tag) and untagged (–Tag) versions of Fc38-63were incubated with anti-V5 antibodies for 30 minutes at 4°C. The cells were thenwashed and lysed. Immune complexes were captured with protein A agarose andsubjected to immunoblotting analysis with V5- or caveolin 1-specific antibodies (B). V5immunoprecipitates prepared from MDCK cells expressing V5-tagged AE1-4 or AE1-4L50A were also processed for immunoblotting analysis with AE1- and caveolin 1-specific antibodies (C). Finally, MDCK cells expressing the wild-type (WT) and mutantV5-tagged Fc38-63 or V5-tagged versions of AE1-4 or AE1-4L50A were lysed in 2 ml ofisotonic buffer containing 1% LubrolWX. The lysates were fractionated on discontinuoussucrose gradients, and 1 ml fractions were collected from the top of the gradient (fraction10 includes the pellet). These samples were either directly analyzed by immunoblottinganalysis using caveolin 1, furin, or �-COP antibodies (D), or immunoprecipitates wereprepared from each fraction using V5-specific antibodies. The immunoprecipitates werethen analyzed by immunoblotting analysis using V5-specific antibodies (D). The top andbottom of the gradient in D are indicated.

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cells expressing the V5-tagged or untagged versions of thechimera were incubated with anti-V5 antibodies for 30 minutesat 4°C. The cells were then washed and lysed, and immunecomplexes were isolated and analyzed by blotting analysis withanti-V5 and anti-caveolin 1 antibodies. This analysis indicatedthat caveolin 1 interacts with the cell surface population of V5-tagged Fc38-63 (Fig. 10B, +Tag).

We also examined whether the endocytosis of Fc38-63 inMDCK cells was dependent upon its ability to partition intodetergent-insoluble lipid rafts. Because we were unable todemonstrate the lipid raft association of caveolin 1 in MDCKcells lysed in a buffer containing Triton X-100, we adopted araft assay protocol (Slimane et al., 2003) in which MDCKcells were lysed in an isotonic buffer containing 1%LubrolWX. Cells lysed in this way were fractionated on adiscontinuous sucrose gradient. Following centrifugation,immunoblotting analysis revealed that the vast majority ofcaveolin 1 floated to positions in the gradient of lighterbuoyant density (fractions 1-6 in Fig. 10D), presumably as aresult of its association with detergent-insoluble lipid rafts.Similar analysis of V5-tagged Fc38-63 revealed that50.2±0.5% (n=2) of the chimeras floated to positions oflighter buoyant density (WT in Fig. 10D). The substitution ofan alanine for the tyrosine or leucine in the YVELtetrapeptide of Fc38-63 did not prevent the lipid raftassociation of the chimera as 31.7±3.7% (n=2) of Fc38-63Y47A and 64.2±0.6% (n=2) of Fc38-63L50A floated topositions of lighter buoyant density in these gradients (Fig.10D). The fact that these mutations inhibited the associationof Fc38-63 with caveolin 1 indicated that the partitioning ofthe chimera into lipid rafts is not dependent upon its abilityto interact with caveolin 1 through its YXX� motif. Blottinganalyses of gradient fractions further revealed that both �-COP, a Golgi marker, and furin, a TGN marker, do not floatto positions of lighter buoyant density in these sucrosegradients (Fig. 10D). These results illustrate that Fc38-63 andfurin, each of which recycle from the plasma membrane tothe Golgi, are not directed to lipid rafts through their YXX�motifs.

Golgi recycling of AE1-4 is linked to its association withcaveolin 1Our previous studies indicated that the chicken AE1-4 anionexchanger acquires its complex N-linked sugar modificationsthrough recycling from the basolateral membrane to the Golgiin polarized MDCK cells (Adair-Kirk et al., 1999). Becauseamino acids 38-63 in our Fc receptor chimeras were derivedfrom the cytoplasmic domain of AE1-4, we also investigatedwhether AE1-4 sorting in MDCK cells was dependent uponits association with caveolin 1. For these analyses, weinserted a V5 epitope tag in the third extracellular loop ofAE1-4. This tag did not alter the sorting or posttranslationalmodification of AE1-4 in MDCK cells (data not shown).Blotting analysis of a V5 immunoprecipitate prepared fromcells expressing V5-tagged AE1-4 revealed that caveolin 1does co-precipitate with this membrane transporter (Fig.10C). To investigate whether the leucine residue in theY47VEL50 sequence of AE1-4 was involved in mediating thisinteraction, it was changed to an alanine. Although V5-taggedAE1-4L50A, like wild-type AE1-4, primarily accumulated inthe basolateral membrane of polarized MDCK cells (Fig.

11A), AE1-4L50A exhibited a more rapid mobility on SDSgels than wild-type AE1-4 (Fig. 10C). Pulse-chaseexperiments revealed that this altered mobility was becauseof the failure of AE1-4L50A to acquire its complex N-linkedsugar modifications (data not shown). In addition to itsaltered mobility on SDS gels, the substitution of an alaninefor leucine 50 in AE1-4 disrupted its association withcaveolin 1 (Fig. 10C). Quantification of two independentexperiments revealed that this substitution resulted in a96.2±3% reduction in caveolin 1 binding to AE1-4. Thesedata strongly suggest that acquisition of mature N-linkedsugars by AE1-4 through recycling from the plasmamembrane to the Golgi is dependent upon its ability toassociate with caveolin 1.

Lipid raft assays investigated whether AE1-4 recycling wasalso dependent upon its ability to partition into detergent-insoluble lipid rafts. These analyses revealed that 54±4.2%(n=2) of AE1-4 associated with lipid raft-containing fractions(Fig. 10D). However, the AE1-4L50A mutant exhibited asimilar capacity to associate with lipid rafts (62±1.5%, n=2).These data indicated that the raft association of AE1-4, likeFc38-63, was not dependent upon caveolin 1 binding.Furthermore, the raft association of AE1-4 is not sufficient toensure its normal trafficking and posttranslational modificationin this epithelial cell type.

Finally, to investigate whether caveolin 1 may be involvedin regulating specific steps in the sorting of AE1-4, weperformed internalization assays using V5 antibodies to labelthe surface population of V5-tagged AE1-4. These analysesrevealed that AE1-4 was retained in the basolateral membraneof MDCK cells that had been incubated at 37°C for 20minutes (Fig. 11B-D). As an alternative approach, weattempted to induce AE1-4 endocytosis using the cholesteroldepletion/repletion protocol used to study the cholesteroldependence of Fc38-63 trafficking (Fig. 4). These analysesrevealed that depleting cellular cholesterol by treating cellswith 10 mM methyl-�-cyclodextrin did not affect thebasolateral localization of AE1-4 (data not shown). However,similar to what was observed for Fc38-63 (Fig. 4), the re-addition of cholesterol to these cells for 20 minutesstimulated AE1-4 to internalize to caveolin 1-positiveendosomes (Fig. 11E-G). The cells shown in Fig. 11E-G wereacid-washed prior to fixation to remove V5 antibodies stillexposed on the surface. If the cells were not acid-washedprior to fixation (Fig. 11H-J), a subset of AE1-4 was stillobserved on the surface following cholesterol repletion. Eventhis surface population of AE1-4 overlapped the distributionof caveolin 1. These data illustrate that some aspects of AE1-4 sorting in MDCK cells are very similar to the resultsobserved for Fc38-63 sorting. Additional control experimentshave shown that the observed accumulation of AE1-4 incaveolin 1-positive endosomes (Fig. 11E-G) does not reflectthe movement of all surface membrane proteins to thiscompartment, because the clathrin-dependent cargos, thelow-density lipoprotein (LDL) receptor and the transferrinreceptor are excluded from caveolin 1-positive endosomesfollowing cholesterol depletion/repletion (data not shown).Collectively, our results point to a crucial role for caveolin 1in regulating the trafficking of a subset of single membrane-spanning and multi-membrane-spanning proteins in thispolarized epithelial cell type.

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DiscussionOur siRNA studies have defined a novel pathway for thecaveolin-dependent endocytosis of a membrane-spanningprotein. These analyses have indicated that the endocytosis ofthe AE1/Fc receptor chimera Fc38-63 is inhibited whencaveolin 1 expression is downregulated in MDCK kidneyepithelial cells. In addition, mutagenesis studies have shownthat the endocytosis of Fc38-63 is dependent upon acytoplasmic YXX� motif that is crucial for the ability of thechimera to associate with caveolin 1. These results representthe first characterization of a cytoplasmic sequence that isnecessary for caveolin-dependent endocytosis. Furthermore,they indicate that YXX� sorting signals, which mediate theclathrin-dependent endocytosis of a variety of membrane

Journal of Cell Science 120 (15)

protein cargo, can also direct caveolin-dependent sorting.Although our data indicate a role for caveolin 1 in regulatingthe endocytosis of Fc38-63, it is unclear at this time whethercaveolin 1 and Fc38-63 are associated during the internalizationstep. It remains possible that the interaction between caveolin1 and Fc38-63 simply serves as a mechanism for delivering thechimera to membrane sub-domains that are competent forendocytosis.

The endocytic pathway of Fc38-63 is most similar to theendocytic pathway of glycosphingolipids, which areinternalized from the plasma membrane of a variety of celltypes in a clathrin-independent and dynamin-dependentfashion (Sharma et al., 2004). Following endocytosis from theplasma membrane, Fc38-63 and glycosphingolipids traversecaveolin 1-positive endosomes prior to being transported to theGolgi (Adair-Kirk et al., 2003; Sharma et al., 2004). However,Fc38-63 is different from glycosphingolipids in that itsendocytosis from the plasma membrane is dependent upon itsability to associate with caveolin 1. Although caveolin 1associates with multiple signaling molecules and cell surfacereceptors (Pingsheng et al., 2002; Cohen et al., 2004), our dataare the first to demonstrate a role for caveolin 1 binding indirecting endocytosis.

Previous analyses have shown that amino acids 38 to 63 inthe cytoplasmic domain of AE1-4 are sufficient to direct plasmamembrane to TGN recycling when fused to a cytoplasmictailless Fc receptor (Adair-Kirk et al., 2003). These sorting dataare consistent with the observation that mutants in Fc38-63 thatare defective in endocytosis have reduced levels of sugarsrelative to the wild-type chimera. The distinct posttranslationalmodifications observed for the wild-type and mutant Fc38-63chimeras may be due exclusively to the different efficiencieswith which these polypeptides undergo endocytosis andrecycling to the Golgi. However, it is also possible that thedifferences in glycosylation arise during the initial transit ofthese polypeptides through the Golgi. Although the precise stepin trafficking that is responsible for the observed differences inposttranslational modification is not known, it is clear thatmutations in the YXX� motif of Fc38-63 that inhibit its abilityto associate with caveolin 1 alter the fate of this chimera inMDCK cells. The YXX�-dependent interaction of Fc38-63 with

Fig. 11. Polarized MDCK cells expressing V5-tagged AE1-4 orAE1-4L50A were fixed and stained with AE1-specific antibodies(A). Alternatively, polarized cells expressing V5-tagged AE1-4 wereincubated at 4°C for 30 minutes in the absence (B-D) or presence (E-J) of 10 mM methyl-�-cyclodextrin. During the 4°C incubation, V5-specific monoclonal antibodies were added to the basolateral surfaceof these polarized cells. The cells were then washed and shifted to37°C for 20 minutes in the absence (B-D) or presence (E-J) of 10mM methyl-�-cyclodextrin/cholesterol. At this time, the cells weredirectly fixed (B-D and H-J) or they were shifted to citrate buffer, pH1.5, for 5 minutes at 4°C to elute surface-bound antibodies prior tofixation (E-G). The cells were then permeabilized and incubated witha rabbit caveolin-1 antibody, followed by anti-mouse IgG conjugatedto Alexa Fluor-594 and anti-rabbit IgG conjugated to Alexa Fluor-488. The xy-slice in each confocal image is approximately in themiddle of the cell with the corresponding xz-slice below. The mergedimages in G and J indicate that AE1-4V5 polypeptides internalizedas a result of cholesterol depletion/repletion accumulate in caveolin1-positive endosomes. Black bars mark the position of the basalmembrane. Bars, 10 �m.

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caveolin 1 is not unique to this membrane-spanning protein. Ourstudies have indicated that caveolin 1 also associates with thechicken AE1-4 anion exchanger, and mutation of leucine 50 inits Y47VEL tetrapeptide disrupts its association with caveolin 1.Interestingly, the AE1-4L50A mutant, which primarily residesin the basolateral membrane of MDCK cells, fails to acquire itscomplex N-linked sugars. These data strongly suggest that therecycling of AE1-4 from the plasma membrane to the Golgi(Adair-Kirk et al., 1999) is dependent upon its association withcaveolin 1.

Depletion of cholesterol by treatment of cells with methyl-�-cyclodextrin dramatically alters the highly organized clustersof cell surface caveolin 1 in certain cell types, such asadipocytes (Kanzaki and Pessin, 2002). However, in contrastto adipocytes, caveolin 1 continued to accumulate in highlyorganized clusters in the basal membrane of methyl-�-cyclodextrin-treated MDCK cells. These clusters overlappedthe distribution of both Fc38-63 (Fig. 6A-C) and AE1-4 (datanot shown). Remarkably, the majority of surface-associatedcaveolin 1 undergoes internalization with Fc38-63 and AE1-4to a common endosomal compartment when cellularcholesterol is repleted by treatment of cells with solublecholesterol. Other investigators have shown that the relativelyimmobile cell surface population of caveolin 1 (Thomsen et al.,2002) can be induced to move following treatment of cells withglycosphingolipids or cholesterol (Sharma et al., 2004). Ourresults further demonstrate that the cholesterol content of theplasma membrane is a crucial determinant for regulating themobility and/or internalization of the surface population ofcaveolin 1 and its associated cargo.

Recent studies have characterized a pathway for theendocytosis of Ctx-B that was dependent upon flotillin 1(Glebov et al., 2006). This raft-associated protein colocalizeswith Ctx-B on the cell surface and in early endosomes, andsiRNA directed against flotillin 1 inhibited Ctx-B endocytosis(Glebov et al., 2006). However, the flotillin-associatedendocytic pathway is dynamin-independent (Glebov et al.,2006), which distinguishes it from the dynamin-dependentendocytic pathway we have characterized for Fc38-63 inMDCK cells.

Previous investigators suggested that caveolin 1 plays anegative rather than a positive role in regulating caveolar-dependent endocytosis (Le et al., 2002). Our siRNA andmutagenesis studies are consistent with a positive regulatory rolefor caveolin 1 in directing Fc38-63 endocytosis in MDCK cells.Additional studies have shown that overexpression of caveolin1 can both positively (Shigematsu et al., 2002) and negatively(Sharma et al., 2004; Kirkham et al., 2005) regulate theendocytosis of cargo. It is difficult to reconcile these variousresults with a single caveolin 1-dependent endocytic pathway.However, it is possible that the recruitment and internalizationof different types of cargo by caveolin 1-dependent pathwayscan be differentially regulated. This differential regulation couldin part be dependent upon whether cargos directly interact withcaveolin 1 or simply reside in membrane microdomains enrichedin caveolin 1.

Materials and MethodsConstruction of siRNA and epitope-tagged constructsA V5 epitope tag was introduced between amino acids 49 and 50 in the extracellulardomain of Fc 38-63 (Adair-Kirk et al., 2003) at an AccIII site in the extracellular

domain of the receptor. In addition, a V5 tag was introduced in the third extracellularloop of AE1-4. The tag was inserted between amino acids 545 and 546 of AE1-4at an AgeI restriction site introduced by site-directed mutagenesis.

Short hairpin RNAs (shRNAs) directed against nucleotides 206-226 of the caninecaveolin 1 and nucleotides 257-277 of EGFP were cloned into the expression vectorpDPEV.neo.

Cell culture and transfectionsTransient transfections were performed using the Effectene transfection reagent(Qiagen). The human transferrin receptor (Grindstaff et al., 1998) and the humanLDL receptor (Gan et al., 2002) were introduced into MDCK cells using areplication-deficient adenovirus.

Transferrin internalization assaysMDCK cells were infected with adenovirus expressing the human transferrinreceptor. One day after infection, the cells were seeded onto Transwell filters(Costar) at confluency. The following day, the cells were transfected with dominant-negative Eps15 (Benmerah et al., 1999), which was fused to EGFP. Forty-eighthours post-transfection, the cells were incubated with human transferrin conjugatedto Alexa Fluor-594 for 30 minutes at 4°C in serum-free Dulbecco’s modified Eagle’smedium (DMEM). The cells were then washed and shifted to 37°C for 30 minutes.The cells were then rinsed and fixed in phosphate-buffered saline (PBS) containing4% paraformaldehyde.

Cholesterol depletion and repletionMDCK cells expressing Fc38-63 were washed with serum-free DMEM andincubated in serum-free DMEM containing 10 mM methyl-�-cyclodextrin for 1hour at 37°C to deplete cholesterol. To replete cholesterol following methyl-�-cyclodextrin treatment, the cells were washed twice with DMEM containing 5%fetal bovine serum and then incubated with 10 mM cholesterol-loaded methyl-�-cyclodextrin (Sigma) at 37°C. Following these treatments, the cells were fixed with4% paraformaldehyde in PBS, and permeabilized by incubation in PBS containing0.5% (vol/vol) Triton X-100 (PBST). The cells were then incubated with the rat2.4G2 anti-Fc receptor monoclonal antibody and affinity-purified rabbit anti-caveolin 1 (Affinity BioReagents) or rabbit anti-furin antibodies (AffinityBioReagents). After washing, cells were incubated with anti-rat IgG conjugated toAlexa Fluor-594 (Molecular Probes) and anti-rabbit IgG conjugated to Alexa Fluor-488 (Molecular Probes).

Polarized MDCK cells expressing V5-tagged AE1-4 grown on permeablesupports were incubated in the absence or presence of 10 mM methyl-�-cyclodextrin for 30 minutes at 4°C. During the 4°C incubation, the basolateralsurface of the cells was incubated with V5-epitope-specific monoclonal antibodies(Invitrogen). The cells were then washed and shifted to 37°C for 20 minutes in theabsence or presence of 10 mM methyl-�-cyclodextrin/cholesterol. At this time, thecells were fixed or they were shifted to 40 mM citric acid, 100 mM KCl, 135 mMNaCl (citrate buffer), pH 1.5, for 5 minutes at 4°C to elute surface-bound antibodiesprior to fixation. The cells were then permeabilized and incubated with a rabbitcaveolin-1 antibody, followed by anti-mouse IgG conjugated to Alexa Fluor-594and anti-rabbit IgG conjugated to Alexa Fluor-488.

Cell surface binding and internalization assaysMDCK cells expressing Fc38-63 were grown on permeable supports. Thebasolateral surface of the cells was incubated with the rat anti-Fc receptor antibody,which recognizes an extracellular epitope of the receptor, for 30 minutes at 4°C.Following washing, the cells were fixed or shifted to pre-warmed media andincubated for 2 or 5 minutes at 37°C. The cells were then incubated in citrate bufferfor 5 minutes at 4°C. Quantification of control experiments revealed that treatmentof cells with citrate buffer prior to shifting the cells to 37°C elutes >99.9% ofsurface-bound antibodies (data not shown). Following the citrate wash, cells wererinsed in PBS, fixed and permeabilized with PBST. The cells were then incubatedwith rabbit antibodies against caveolin 1, clathrin (Sigma) or early endosomalantigen (EEA1, Affinity BioReagents) followed by the appropriate secondaryantibodies. Z-stacks were collected through the cells using a Zeiss LSM510 confocalmicroscope. The percentage of pixels resulting from Fc38-63 staining thatcolocalized with the various markers in each slice in the z-stack was determinedusing the colocalization function in the Zeiss software.

In some instances, cells were simultaneously incubated with the rat anti-Fc

receptor antibody and transferrin conjugated to Alexa Fluor-488 (Molecular Probes)or Ctx-B conjugated to Alexa Fluor-488 (Molecular Probes) for 30 minutes at 4°C.After washing, the cells were either fixed or shifted to 37°C for 2 or 5 minutes.Following the 37°C incubation, the cells were incubated in citrate buffer for 5minutes at 4°C to elute surface-bound molecules. The cells were then fixed,permeabilized and incubated with anti-rat IgG conjugated to Alexa Fluor-594. Thepercentage of pixels resulting from Fc38-63 staining in each slice of a z-stack thatcolocalized with transferrin or Ctx-B was determined.

In some experiments, Fc38-63-expressing cells were transfected with wild-typedynamin 2, dominant-negative dynamin 2 (K44A) (Altschuler et al., 1998) or withdominant-negative Eps15�95-295. The dynamin cDNAs were cloned in

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pDPEV.neo-EGFP, which encoded EGFP under the control of a separate promoter.Forty-eight hours post-transfection, the cells were incubated with the rat anti-Fc

receptor antibody for 30 minutes at 4°C. Following washing, the cells wereincubated at 37°C for 30 minutes. The cells were then fixed and processed asdescribed above.

Fc38-63-expressing cells were also transfected with the caveolin 1 or EGFPshRNA-expressing plasmids. Forty-eight hours post-transfection, the cells wereincubated with the anti-Fc receptor antibody for 30 minutes at 4°C. Followingwashing, the cells were shifted to 37°C for 2 minutes. At this time, the cells wereincubated in citrate buffer, pH 1.5, for 5 minutes at 4°C to elute surface antibodies.The cells were then fixed, permeabilized and incubated with secondary antibody.Following washing, z-stacks were collected through the cells and the effect of theseconstructs on Fc38-63 endocytosis was determined by comparing the total numberof pixels in transfected cells with those present in neighboring cells that were non-transfected. Similar analyses investigated the effect of these reagents on transferrinand Ctx-B endocytosis.

Immunoprecipitation and immunoblottingMDCK cells expressing V5-tagged versions of Fc38-63, Fc38-63Y47A, Fc38-63L50A, AE1-4 or AE1-4L50A were lysed in isotonic buffer containing 1%(vol/vol) Triton X-100 and processed for immunoprecipitation as describedpreviously (Adair-Kirk et al., 1999) using anti-V5 antibodies. In some instances,precipitates were incubated with a glycosidase mixture (Calbiochem) that removesboth O-linked and N-linked sugars. Alternatively, MDCK cells expressing untaggedand V5-tagged versions of Fc38-63 were incubated with anti-V5 antibodies for 30minutes at 4°C. The cells were then washed and lysed as described above, andimmune complexes were isolated using protein A agarose. Immunoprecipitatesprepared in both ways were subjected to immunoblotting analysis using themonoclonal anti-V5 antibody.

Lipid raft assaysMDCK cells expressing V5-tagged versions of Fc38-63, Fc38-63Y47A, Fc38-63L50A, AE1-4 or AE1-4L50A were rinsed with PBS and lysed in 2 ml of 20 mMTris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA containing 1% LubrolWX at 4°C(Slimane et al., 2003). The lysates were mixed with an equal volume of 80% sucrosein the same buffer lacking LubrolWX, and overlaid with a discontinuous sucrosegradient (5 ml of 30% sucrose and 1 ml of 5% sucrose in buffer lacking LubrolWX).The samples were centrifuged at 200,000 g for 20 hours, and 1 ml fractions werecollected from the top of the gradient and analyzed by immunoblotting analysis.Alternatively, immunoprecipitates were prepared from each fraction using V5-specific antibodies and subsequently analyzed by blotting analysis.

This research was supported by a grant from the Southeast Affiliateof the American Heart Association (0355333B). Dynamin cDNAswere provided by D. Vignali. Adenoviral vectors expressing thetransferrin and LDL receptors were provided by I. Mellman and E.Rodriguez-Boulan, respectively. Eps15 cDNAs were provided by A.Benmerah. We thank K. Cox and P. Ryan for their critical evaluationof the manuscript.

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