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First published online 10 July 2007
doi: 10.1242/jcs.002493

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A novel role for a YXX motif in directing the caveolin-dependent sorting of membrane-spanning proteins

Frank 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

Author for correspondence (e-mail: jcox{at}utmem.edu)

Accepted 21 May 2007

	Summary

Top Summary Introduction Results Discussion Materials and Methods References

 
Previous studies showed that the sequence between amino acids 38 and 63 of the chicken AE1-4 anion exchanger is sufficient to direct basolateral sorting and recycling to the Golgi when fused to a cytoplasmic tailless F_cRII B2 receptor. Further characterization of the recycling pathway has indicated that the chimera F_c38-63 colocalizes with caveolin 1 in the basolateral membrane of MDCK cells, and in early endosomes following its internalization from the cell surface. Studies using small interfering RNA (siRNA) and dominant-negative mutants revealed that F_c38-63 endocytosis is primarily caveolin-dependent and clathrin-independent. The endocytosis of the chimera is also dependent upon cholesterol and dynamin. Co-precipitation studies indicated that caveolin 1 associates with F_c38-63. Mutation of the tyrosine or leucine residues in the cytoplasmic sequence Y₄₇VEL of F_c38-63 disrupts this interaction and inhibits the endocytosis of the chimera. Additional analyses revealed that AE1-4 also associates with caveolin 1. Mutation of the leucine in the Y₄₇VEL sequence of AE1-4 disrupts this interaction, and blocks the recycling of this transporter from the basolateral membrane to the Golgi. The Y₄₇VEL tetrapeptide matches the sequence of a YXX motif, and our results indicate a novel role for this motif in directing caveolin-dependent sorting.

Key words: Caveolin, Sorting signal, Endosomes, AE1 anion exchanger

	Introduction

Top Summary Introduction Results Discussion Materials and Methods References

 
Multiple sorting signals that reside at the N-terminus of the cytoplasmic domain of the chicken kidney AE1-4 anion exchanger direct the basolateral sorting and Golgi recycling of this membrane transporter in polarized MDCK cells (Adair-Kirk et al., 1999; Adair-Kirk et al., 2003). Recent studies have shown that one of these signals, which is located between amino acids 38 and 63 of AE1-4, is sufficient to direct basolateral sorting when fused to a cytoplasmic tailless murine IgG F_cRII B2 receptor. However, this chimeric receptor, F_c38-63, primarily accumulates in the trans-Golgi-network (TGN) at steady state. This localization profile is dependent upon recycling of F_c38-63 from the basolateral membrane to the TGN. Mutagenesis studies have shown that the TGN recycling of F_c38-63 is dependent upon a tyrosine residue located in the tetrapeptide Y₄₇VEL, which matches the sequence of a YXX motif, where X is any amino acid and is a hydrophobic residue. This motif directs the clathrin-dependent endocytosis of membrane-spanning proteins (Collawn et al., 1990) through its association with the AP2 clathrin adaptor complex (Aguilar et 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 and their internalization from the cell surface requires the tyrosine-based endocytic signals YKGL and YQRL, respectively. The endocytic 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 the hydrophobic residue in their YXX motifs (Humphrey et al., 1993; Owen and Evans, 1998).

Some lipids (Sharma et al., 2003; Singh et al., 2003) and proteins (Le et al., 2002) that recycle from the plasma membrane to early compartments of the secretory pathway are internalized by non-clathrin-dependent endocytic pathways. Cargo internalized by clathrin-independent pathways often traverse caveolin 1-positive endosomes prior to their delivery to early secretory pathway compartments (Pelkmans et al., 2001; Nichols, 2002; Sharma et al., 2004). However, the endocytosis of these clathrin-independent cargos from the plasma membrane can occur through vesicular carriers coated with 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 recycling pathway of F_c38-63 in MDCK cells. Studies using small interfering RNA (siRNA) and dominant-negative mutants show that even though the endocytosis of F_c38-63 is dependent upon a YXX motif, it is internalized from the plasma membrane through a caveolin-dependent pathway. Moreover, co-precipitation studies indicate that caveolin 1 is associated with F_c38-63 and the AE1-4 anion exchanger in this kidney epithelial cell type. Mutations in the cytoplasmic Y₄₇VEL tetrapeptide of these proteins disrupt their interaction with caveolin 1. The fact that these mutations also inhibit F_c38-63 and AE1-4 trafficking suggests a novel role for YXX motifs in targeting membrane-spanning proteins to caveolin-dependent sorting pathways.

	Results

Top Summary Introduction Results Discussion Materials and Methods References

 
Eps15-independent endocytosis of F_c38-63 is dependent upon a YXX motif
Previous studies using chimeras between the chicken AE1-4 anion exchanger and a cytoplasmic tailless murine IgG F_cRII B2 receptor defined multiple sorting signals within the cytoplasmic domain of AE1-4 (Adair-Kirk et al., 1999; Adair-Kirk et al., 2003). One of these signals, which resides between amino acids 38 and 63 of AE1-4, was sufficient to direct the basolateral sorting and recycling of the AE1/F_c receptor chimera F_c38-63 from the plasma membrane to the TGN. Mutagenesis studies demonstrated that the steady-state localization of this chimera was altered when the tyrosine or leucine residues located in the sequence Y₄₇VEL₅₀ were changed to an alanine. This tetrapeptide matches the sequence of 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 the Y₄₇VEL₅₀ sequence of F_c38-63 were involved in directing the endocytosis of this chimera, MDCK cells expressing wild-type F_c38-63, F_c38-63L50A or F_c38-63Y47A were incubated with an antibody that recognizes an extracellular epitope on the F_c receptor for 30 minutes at 4°C to prevent endocytosis and then shifted to 37°C for 45 minutes. As shown previously (Adair-Kirk et al., 2003), wild-type F_c38-63 internalized from the cell surface and accumulated in a perinuclear compartment of MDCK cells (Fig. 1A-C). By contrast, both F_c38-63L50A (Fig. 1D-F) and F_c38-63Y47A (Fig. 1G-I) were almost entirely retained in the basolateral membrane of these epithelial cells, indicating a crucial role for these residues in directing endocytosis.

View larger version (151K): [in this window] [in a new window]   Fig. 1. MDCK cells expressing Fc38-63 (A-C), Fc38-63L50A (D-F) or Fc38-63Y47A (G-I) were incubated with rat anti-Fc receptor antibodies for 30 minutes at 4°C and then shifted to 37°C for 45 minutes. Cells were then fixed, permeabilized and incubated with anti-rat IgG conjugated to Alexa Fluor-594 (A,D,G) and Alexa Fluor-488-phalloidin (B,E,H). The confocal images correspond to an xy-slice through the middle of the cells. Bars, 10 µm.

View larger version (37K): [in this window] [in a new window]   Fig. 2. MDCK cells expressing Fc38-63 or the human transferrin receptor were transfected with EGFP-tagged Eps1595-295 (B,E). The cells were incubated with Alexa Fluor-594-transferrin (A) or anti-Fc receptor antibodies (D) for 30 minutes at 4°C and then shifted to 37°C for 30 minutes. Cells were then fixed and immunofluorescent molecules were directly visualized (A-C) or the cells were fixed, permeabilized and incubated with anti-rat IgG conjugated to Alexa Fluor-594 prior to microscopic analysis (D-F). Bars, 10 µm.

View larger version (69K): [in this window] [in a new window]   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 and incubated with rat anti-Fc-receptor antibody and with a rabbit antibody directed against furin. After washing, cells were incubated with anti-rat IgG conjugated to Alexa Fluor-594 and anti-rabbit IgG conjugated to Alexa Fluor-488. Alternatively, after a 1-hour incubation in 10 mM methyl--cyclodextrin, the cells were washed with DMEM containing 5% fetal bovine serum and incubated for 20 minutes (G-I) or 60 minutes (J-L) with 10 mM methyl--cyclodextrin loaded with cholesterol and processed as described above. The upper panel in each confocal image corresponds to an xy-slice near the middle of the cells, whereas the lower panel corresponds to an xz-slice. Black bars mark the position of the basal membrane. Bars, 10 µm.

View larger version (69K): [in this window] [in a new window]   Fig. 4. MDCK cells expressing Fc38-63 were processed as described in the legend to Fig. 3 except the cells were incubated with caveolin 1 antibodies rather than antibodies to furin. The upper panel in each confocal image corresponds to an xy-slice near the middle of the cells, whereas the lower panel corresponds to an xz-slice. Black bars mark the position of the basal membrane. Bars, 10 µm.

Cellular cholesterol was replenished in methyl--cyclodextrin-treated cells by incubating the cells in the presence of soluble cholesterol/methyl--cyclodextrin complexes. Treatment of cholesterol-depleted MDCK cells in this way stimulated the internalization of the basolateral population of F_c38-63. Within 20 minutes of adding cholesterol to the media, the surface population of F_c38-63 and caveolin 1 internalized, and these proteins were colocalized in endosomes (Fig. 4G-I). Additional studies revealed that the majority of the surface chimeras internalized within 3 minutes of adding cholesterol to the media (data not shown). One hour after the addition of cholesterol, some of the chimeras still overlapped the distribution of caveolin 1 (Fig. 4J-L). However, a significant fraction of F_c38-63 assumed a TGN localization profile where it again overlapped the distribution of furin (Fig. 3J-L). These data indicated that under the conditions of this cholesterol depletion/repletion protocol, some of the steps in the trafficking of F_c38-63 in MDCK cells were linked to the trafficking of caveolin 1.

The caveolar-dependent endocytosis of glycosphingolipids is dependent upon dynamin (Puri et al., 2001), which is necessary for the pinching off of caveolae from the plasma membrane. To determine whether F_c38-63 endocytosis exhibited a similar dynamin-dependence, MDCK cells expressing the chimera were transfected with the dominant-negative dynamin 2(K44A) mutant. The transfected cells were incubated with the F_c receptor antibody for 30 minutes at 4°C and then shifted to 37°C for 30 minutes. This analysis revealed that the internalization of F_c38-63 was almost entirely inhibited in cells expressing dominant-negative dynamin (Fig. 5D-F), whereas similar studies with wild-type dynamin had no effect on chimera uptake (Fig. 5A-C). Taken together, our data unexpectedly showed that F_c38-63 was internalized through a pathway that was cholesterol- and dynamin-dependent and Eps15-independent. These properties are hallmarks of caveolar-dependent endocytosis. The fact that F_c38-63 internalization was also dependent upon its YXX motif suggested a novel role for this sorting signal in directing an endocytic pathway linked to caveolin 1.

View larger version (51K): [in this window] [in a new window]   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 a vector that also expresses EGFP. In each instance, the cells were incubated with anti-Fc receptor antibodies for 30 minutes at 4°C and then shifted to 37°C for 30 minutes. Cells were then fixed, permeabilized and incubated with anti-rat IgG conjugated to Alexa Fluor-594. Bars, 10 µm.

View larger version (120K): [in this window] [in a new window]   Fig. 6. Polarized MDCK cells expressing Fc38-63 were incubated in the presence of 10 mM methyl--cyclodextrin for 1 hour at 37°C. The cells were then fixed, permeabilized and incubated with antibodies specific for the Fc receptor (A) and caveolin 1 (B). Alternatively, untreated cells were incubated with anti-Fc receptor antibodies (D-F) for 30 minutes at 4°C. The cells were then washed, fixed, permeabilized and incubated with antibodies specific for caveolin 1 (E). In both instances, the cells were washed and incubated with anti-rat IgG conjugated to Alexa Fluor-594 (A,D) and anti-rabbit IgG conjugated to Alexa Fluor-488 (B,E). Bars, 10 µm.

View larger version (52K): [in this window] [in a new window]   Fig. 7. Polarized MDCK cells expressing Fc38-63 were grown on permeable supports. The basolateral surface of the cells was incubated with anti-Fc receptor antibodies (A) for 30 minutes at 4°C. The cells were then washed and shifted to 37°C for 2 minutes. After 2 minutes of incubation, the cells were shifted to citrate buffer, pH 1.5, for 5 minutes at 4°C to elute surface-bound antibodies. The cells were then rinsed in PBS and fixed. Following permeabilization, the cells were incubated with antibodies specific for caveolin 1 (B). The cells were again washed and incubated with anti-rat IgG conjugated to Alexa Fluor-594 (A) and anti-rabbit IgG conjugated to Alexa Fluor-488 (B). The merged image (C) illustrates the overlap between the chimera and caveolin. The xy-slice in each confocal image is approximately 1 µm above the basal membrane of the cell. Similar analyses have quantified the colocalization of Fc38-63 with clathrin, transferrin, Ctx-B or EEA1. The bar graph in D shows the percentage of the total number of pixels resulting from Fc38-63 staining that colocalized with these various endocytic markers on the cell surface (0 Min.), and at 2 and 5 minutes post-internalization. This quantification reflects the average result from at least 25 cells from two independent experiments. Bar, 1 µm.

Shifting the cells to 37°C for 2 minutes induced the endocytosis of the chimeras. The higher magnification images in Fig. 7A-C illustrate that a substantial fraction of the internalized chimeras accumulate in caveolin 1-positive endosomes. Quantification of this assay revealed that 61% of the internalized chimeras accumulated in this caveolin 1-positive endosomal compartment. Additional quantitative analyses revealed that 33% of internalized F_c38-63 accumulated in endosomes containing Ctx-B that had also been endocytosed from the cell surface (Fig. 7D), whereas only 8% accumulated in endosomes containing internalized transferrin (Fig. 7D). The percentage of F_c38-63 that accumulated in transferrin-positive endosomes was identical to the percentage that colocalized with clathrin on the cell surface (Fig. 7D), suggesting that a small percentage of the chimeras are internalized by clathrin-dependent endocytosis and delivered to transferrin-positive endosomes. Twenty percent of internalized F_c38-63 was present in endosomes that were positive for EEA1 (Fig. 7D), a marker for early endosomes that acquire cargo internalized by clathrin and caveolar-dependent endocytosis (Pelkmans et al., 2004). The fact that internalized F_c38-63 minimally overlapped the distribution of Ctx-B, transferrin and EEA1, which are markers for both clathrin- and caveolar-dependent endocytosis, suggested the chimeras are endocytosed by a novel pathway. Five minutes after the shift to 37°C, the percentage of internalized chimeras present in endosomes containing caveolin 1 and Ctx-B increased slightly to 73% and 53%, respectively, whereas the chimeras present in endosomes containing transferrin and EEA1 were essentially unchanged (Fig. 7D). The increased overlap between F_c38-63 and Ctx-B at the 5-minute time point suggests that these cargo transit through a common caveolin 1-positive endosomal compartment prior to their delivery to early compartments of the secretory pathway (Adair-Kirk et al., 2003; Nichols, 2002).

F_c38-63 endocytosis is caveolin 1-dependent
To determine whether caveolin 1 is involved in the endocytosis of F_c38-63, an siRNA strategy was employed to downregulate caveolin 1 expression in MDCK cells. For these analyses, we transiently expressed a caveolin 1 siRNA in MDCK cells using a vector that expressed EGFP under the control of a separate promoter. The sequence used for the siRNA has been used by other investigators to downregulate caveolin 1 expression in this cell type (Manninen et al., 2005). Immunolocalization studies revealed that caveolin 1 expression levels were substantially reduced in cells expressing high levels of the EGFP reporter (Fig. 8A-C). Quantification of images similar to this revealed that the caveolin 1 siRNA reduced caveolin 1 expression levels to 35% of that observed in non-transfected cells (Fig. 8G). The caveolin remaining in transfected cells probably represents a stable population of caveolin that has been observed in previous studies in which this same siRNA was used to downregulate caveolin expression (Schuck et al., 2004). Additional studies using a control siRNA directed against EGFP had no effect on caveolin 1 expression (data not shown).

View larger version (55K): [in this window] [in a new window]   Fig. 8. Subconfluent MDCK cells expressing Fc38-63 were transfected with a vector expressing the caveolin 1 siRNA. This vector also expressed EGFP under the control of a separate promoter. Forty-eight hours post-transfection, the cells were fixed (A-C) or incubated with anti-Fc receptor antibodies for 30 minutes at 4°C, washed and then fixed (D-F). Following permeabilization, the cells in A-C were incubated with caveolin 1 antibodies (A) followed by anti-rabbit IgG conjugated to Alexa Fluor-594. The cells in D-F were directly 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 the caveolin 1 siRNA. Note the absence of caveolin staining in the plasma membrane. The bar graph in G has quantified the average effect of the caveolin 1 siRNA on the caveolin 1 protein level and Fc38-63 surface expression in 20 randomly chosen cells from two independent experiments. Bars, 10 µm.

View larger version (45K): [in this window] [in a new window]   Fig. 9. Subconfluent MDCK cells expressing Fc38-63 were transfected with the caveolin 1 siRNA-expressing vector. Forty-eight hours post-transfection, the cells were incubated with anti-Fc receptor antibodies, Ctx-B conjugated to Alexa Fluor-594 or transferrin conjugated to Alexa Fluor-594 for 30 minutes at 4°C. The cells were then washed and shifted to 37°C for 2 minutes. At this time, the cells were shifted to citrate buffer, pH 1.5, for 5 minutes at 4°C to elute surface-bound molecules. The cells were then rinsed in PBS and fixed. Following permeabilization, cells that had been incubated with anti-Fc receptor antibodies were incubated with anti-rat IgG conjugated to Alexa Fluor-594. Immunofluorescent molecules were visualized on a Zeiss LSM 510 confocal microscope. The bar graph in B has quantified the effect of the caveolin 1 siRNA on the endocytosis of Fc38-63 and transferrin in 20 randomly chosen cells. The effect of a control siRNA directed against EGFP on Fc38-63 endocytosis is also illustrated (B). The standard error in B is indicated. Bars, 10 µm.

Association of F_c38-63 with caveolin 1 is dependent upon its YXX motif
The data described above indicated that F_c38-63 endocytosis is significantly impaired when caveolin 1 expression is downregulated. To investigate whether an interaction between F_c38-63 and caveolin 1 may be necessary for the endocytosis of the chimera, co-precipitation studies were performed. Because we were unable to immunoprecipitate F_c38-63 using the anti-F_c receptor antibody, we inserted a V5 epitope tag in the extracellular domain of F_c38-63. Localization and internalization assays with V5-tagged F_c38-63 revealed that the epitope tag did not alter the basolateral sorting of the chimera. Although the kinetics of endocytosis of V5-tagged F_c38-63 were slightly slower than the untagged chimera, V5-tagged F_c38-63 traversed the same compartments as the untagged chimera during recycling to the Golgi (data not shown). Immunoblotting analysis of a V5 immunoprecipitate prepared from cells expressing V5-tagged F_c38-63 revealed that the chimera migrated as a diffuse species ranging in size from 47 kDa to 50 kDa (Fig. 10A). Similar blotting analysis of V5-tagged F_c38-63Y47A and F_c38-63L50A indicated that these amino acid substitutions, which inhibited the endocytosis of the chimera, also altered the mobility of the proteins on sodium dodecyl sulfate (SDS) gels. F_c38-63Y47A migrated as two species of 42 kDa and 45 kDa, whereas F_c38-63L50A primarily migrated as two species of 45 kDa and 47 kDa (Fig. 10A). To address the possibility that the protein profiles observed in this blotting analysis reflected heterogeneity in the acquisition of sugars on the four potential N-linked glycosylation sites of the chimeras, the V5 immunoprecipitates were digested with glycosidases to remove all sugars prior to blotting analysis. This experiment revealed that each chimera migrated as two species of 32 kDa and 35 kDa following glycosidase digestion (Fig. 10A). These species were slightly larger than the predicted molecular mass of these proteins, which was 29 kDa. Whether the 32 kDa or 35 kDa species possess modifications in addition to glycosylation is not known at this time. However, these data clearly indicate that the heterogeneity observed for the wild-type and mutant chimeras was because of variability in sugar acquisition. The reduced level of sugar addition on F_c38-63Y47A and F_c38-63L50A relative to the wild-type chimera correlated with the reduced ability of the mutants to undergo endocytosis and recycling to the Golgi following surface delivery.

View larger version (47K): [in this window] [in a new window]   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 of glycosidases that removes all O-linked and N-linked sugars prior to immunoblotting analysis with V5 antibodies (A). V5 immunoprecipitates prepared from MDCK cells expressing V5-tagged versions of Fc38-63, Fc38-63Y47A, Fc38-63L50A were also subjected to immunoblotting analysis with V5- and caveolin 1-specific antibodies (B). Alternatively, cells expressing V5-tagged (+Tag) and untagged (–Tag) versions of Fc38-63 were incubated with anti-V5 antibodies for 30 minutes at 4°C. The cells were then washed and lysed. Immune complexes were captured with protein A agarose and subjected to immunoblotting analysis with V5- or caveolin 1-specific antibodies (B). V5 immunoprecipitates 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 mutant V5-tagged Fc38-63 or V5-tagged versions of AE1-4 or AE1-4L50A were lysed in 2 ml of isotonic buffer containing 1% LubrolWX. The lysates were fractionated on discontinuous sucrose gradients, and 1 ml fractions were collected from the top of the gradient (fraction 10 includes the pellet). These samples were either directly analyzed by immunoblotting analysis using caveolin 1, furin, or -COP antibodies (D), or immunoprecipitates were prepared from each fraction using V5-specific antibodies. The immunoprecipitates were then analyzed by immunoblotting analysis using V5-specific antibodies (D). The top and bottom of the gradient in D are indicated.

To determine whether F_c38-63 associated with caveolin 1, V5 immunoprecipitates prepared from MDCK cells transfected with V5-tagged F_c38-63 were probed with caveolin 1 antibodies. This analysis revealed that caveolin 1 coprecipitated with V5-tagged F_c38-63 (Fig. 10B). Similar studies using cells transfected with V5-tagged F_c38-63Y47A and F_c38-63L50A indicated that these amino acid substitutions almost completely blocked the association of caveolin 1 with the chimera (Fig. 10B). Quantification of three independent experiments revealed that the Y47A substitution resulted in a 99.2±1.4% reduction in the amount of caveolin 1 that coprecipitated with V5-tagged F_c38-63, whereas the L50A substitution resulted in a 97.9±3.6% reduction. The fact that these mutant chimeras are also defective in endocytosis suggested that the YXX-dependent interaction of the chimera with caveolin 1 is necessary for its endocytosis from the plasma membrane. To ensure that the surface population of F_c38-63 does indeed associate with caveolin 1, MDCK cells expressing the V5-tagged or untagged versions of the chimera were incubated with anti-V5 antibodies for 30 minutes at 4°C. The cells were then washed and lysed, and immune complexes were isolated and analyzed by blotting analysis with anti-V5 and anti-caveolin 1 antibodies. This analysis indicated that caveolin 1 interacts with the cell surface population of V5-tagged F_c38-63 (Fig. 10B, +Tag).

We also examined whether the endocytosis of F_c38-63 in MDCK cells was dependent upon its ability to partition into detergent-insoluble lipid rafts. Because we were unable to demonstrate the lipid raft association of caveolin 1 in MDCK cells lysed in a buffer containing Triton X-100, we adopted a raft assay protocol (Slimane et al., 2003) in which MDCK cells were lysed in an isotonic buffer containing 1% LubrolWX. Cells lysed in this way were fractionated on a discontinuous sucrose gradient. Following centrifugation, immunoblotting analysis revealed that the vast majority of caveolin 1 floated to positions in the gradient of lighter buoyant density (fractions 1-6 in Fig. 10D), presumably as a result of its association with detergent-insoluble lipid rafts. Similar analysis of V5-tagged F_c38-63 revealed that 50.2±0.5% (n=2) of the chimeras floated to positions of lighter buoyant density (WT in Fig. 10D). The substitution of an alanine for the tyrosine or leucine in the YVEL tetrapeptide of F_c38-63 did not prevent the lipid raft association of the chimera as 31.7±3.7% (n=2) of F_c38-63Y47A and 64.2±0.6% (n=2) of F_c38-63L50A floated to positions of lighter buoyant density in these gradients (Fig. 10D). The fact that these mutations inhibited the association of F_c38-63 with caveolin 1 indicated that the partitioning of the chimera into lipid rafts is not dependent upon its ability to interact with caveolin 1 through its YXX motif. Blotting analyses of gradient fractions further revealed that both -COP, a Golgi marker, and furin, a TGN marker, do not float to positions of lighter buoyant density in these sucrose gradients (Fig. 10D). These results illustrate that F_c38-63 and furin, each of which recycle from the plasma membrane to the Golgi, are not directed to lipid rafts through their YXX motifs.

Golgi recycling of AE1-4 is linked to its association with caveolin 1
Our previous studies indicated that the chicken AE1-4 anion exchanger acquires its complex N-linked sugar modifications through recycling from the basolateral membrane to the Golgi in polarized MDCK cells (Adair-Kirk et al., 1999). Because amino acids 38-63 in our F_c receptor chimeras were derived from the cytoplasmic domain of AE1-4, we also investigated whether AE1-4 sorting in MDCK cells was dependent upon its association with caveolin 1. For these analyses, we inserted a V5 epitope tag in the third extracellular loop of AE1-4. This tag did not alter the sorting or posttranslational modification of AE1-4 in MDCK cells (data not shown). Blotting analysis of a V5 immunoprecipitate prepared from cells expressing V5-tagged AE1-4 revealed that caveolin 1 does co-precipitate with this membrane transporter (Fig. 10C). To investigate whether the leucine residue in the Y₄₇VEL₅₀ sequence of AE1-4 was involved in mediating this interaction, it was changed to an alanine. Although V5-tagged AE1-4L50A, like wild-type AE1-4, primarily accumulated in the basolateral membrane of polarized MDCK cells (Fig. 11A), AE1-4L50A exhibited a more rapid mobility on SDS gels than wild-type AE1-4 (Fig. 10C). Pulse-chase experiments revealed that this altered mobility was because of the failure of AE1-4L50A to acquire its complex N-linked sugar modifications (data not shown). In addition to its altered mobility on SDS gels, the substitution of an alanine for leucine 50 in AE1-4 disrupted its association with caveolin 1 (Fig. 10C). Quantification of two independent experiments revealed that this substitution resulted in a 96.2±3% reduction in caveolin 1 binding to AE1-4. These data strongly suggest that acquisition of mature N-linked sugars by AE1-4 through recycling from the plasma membrane to the Golgi is dependent upon its ability to associate with caveolin 1.

View larger version (52K): [in this window] [in a new window]   Fig. 11. Polarized MDCK cells expressing V5-tagged AE1-4 or AE1-4L50A were fixed and stained with AE1-specific antibodies (A). Alternatively, polarized cells expressing V5-tagged AE1-4 were incubated 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 surface of these polarized cells. The cells were then washed and shifted to 37°C for 20 minutes in the absence (B-D) or presence (E-J) of 10 mM methyl--cyclodextrin/cholesterol. At this time, the cells were directly fixed (B-D and H-J) or they were shifted to citrate buffer, pH 1.5, for 5 minutes at 4°C to elute surface-bound antibodies prior to fixation (E-G). The cells were then permeabilized and incubated with a rabbit caveolin-1 antibody, followed by anti-mouse IgG conjugated to Alexa Fluor-594 and anti-rabbit IgG conjugated to Alexa Fluor-488. The xy-slice in each confocal image is approximately in the middle of the cell with the corresponding xz-slice below. The merged images in G and J indicate that AE1-4V5 polypeptides internalized as a result of cholesterol depletion/repletion accumulate in caveolin 1-positive endosomes. Black bars mark the position of the basal membrane. Bars, 10 µm.

Finally, to investigate whether caveolin 1 may be involved in regulating specific steps in the sorting of AE1-4, we performed internalization assays using V5 antibodies to label the surface population of V5-tagged AE1-4. These analyses revealed that AE1-4 was retained in the basolateral membrane of MDCK cells that had been incubated at 37°C for 20 minutes (Fig. 11B-D). As an alternative approach, we attempted to induce AE1-4 endocytosis using the cholesterol depletion/repletion protocol used to study the cholesterol dependence of F_c38-63 trafficking (Fig. 4). These analyses revealed that depleting cellular cholesterol by treating cells with 10 mM methyl--cyclodextrin did not affect the basolateral localization of AE1-4 (data not shown). However, similar to what was observed for F_c38-63 (Fig. 4), the re-addition of cholesterol to these cells for 20 minutes stimulated AE1-4 to internalize to caveolin 1-positive endosomes (Fig. 11E-G). The cells shown in Fig. 11E-G were acid-washed prior to fixation to remove V5 antibodies still exposed on the surface. If the cells were not acid-washed prior to fixation (Fig. 11H-J), a subset of AE1-4 was still observed on the surface following cholesterol repletion. Even this surface population of AE1-4 overlapped the distribution of caveolin 1. These data illustrate that some aspects of AE1-4 sorting in MDCK cells are very similar to the results observed for F_c38-63 sorting. Additional control experiments have shown that the observed accumulation of AE1-4 in caveolin 1-positive endosomes (Fig. 11E-G) does not reflect the movement of all surface membrane proteins to this compartment, because the clathrin-dependent cargos, the low-density lipoprotein (LDL) receptor and the transferrin receptor are excluded from caveolin 1-positive endosomes following cholesterol depletion/repletion (data not shown). Collectively, our results point to a crucial role for caveolin 1 in regulating the trafficking of a subset of single membrane-spanning and multi-membrane-spanning proteins in this polarized epithelial cell type.

	Discussion

Top Summary Introduction Results Discussion Materials and Methods References

 
Our siRNA studies have defined a novel pathway for the caveolin-dependent endocytosis of a membrane-spanning protein. These analyses have indicated that the endocytosis of the AE1/F_c receptor chimera F_c38-63 is inhibited when caveolin 1 expression is downregulated in MDCK kidney epithelial cells. In addition, mutagenesis studies have shown that the endocytosis of F_c38-63 is dependent upon a cytoplasmic YXX motif that is crucial for the ability of the chimera to associate with caveolin 1. These results represent the first characterization of a cytoplasmic sequence that is necessary for caveolin-dependent endocytosis. Furthermore, they indicate that YXX sorting signals, which mediate the clathrin-dependent endocytosis of a variety of membrane protein cargo, can also direct caveolin-dependent sorting. Although our data indicate a role for caveolin 1 in regulating the endocytosis of F_c38-63, it is unclear at this time whether caveolin 1 and F_c38-63 are associated during the internalization step. It remains possible that the interaction between caveolin 1 and F_c38-63 simply serves as a mechanism for delivering the chimera to membrane sub-domains that are competent for endocytosis.

The endocytic pathway of F_c38-63 is most similar to the endocytic pathway of glycosphingolipids, which are internalized from the plasma membrane of a variety of cell types in a clathrin-independent and dynamin-dependent fashion (Sharma et al., 2004). Following endocytosis from the plasma membrane, F_c38-63 and glycosphingolipids traverse caveolin 1-positive endosomes prior to being transported to the Golgi (Adair-Kirk et al., 2003; Sharma et al., 2004). However, F_c38-63 is different from glycosphingolipids in that its endocytosis from the plasma membrane is dependent upon its ability to associate with caveolin 1. Although caveolin 1 associates with multiple signaling molecules and cell surface receptors (Pingsheng et al., 2002; Cohen et al., 2004), our data are the first to demonstrate a role for caveolin 1 binding in directing endocytosis.

Previous analyses have shown that amino acids 38 to 63 in the cytoplasmic domain of AE1-4 are sufficient to direct plasma membrane to TGN recycling when fused to a cytoplasmic tailless F_c receptor (Adair-Kirk et al., 2003). These sorting data are consistent with the observation that mutants in F_c38-63 that are defective in endocytosis have reduced levels of sugars relative to the wild-type chimera. The distinct posttranslational modifications observed for the wild-type and mutant F_c38-63 chimeras may be due exclusively to the different efficiencies with which these polypeptides undergo endocytosis and recycling to the Golgi. However, it is also possible that the differences in glycosylation arise during the initial transit of these polypeptides through the Golgi. Although the precise step in trafficking that is responsible for the observed differences in posttranslational modification is not known, it is clear that mutations in the YXX motif of F_c38-63 that inhibit its ability to associate with caveolin 1 alter the fate of this chimera in MDCK cells. The YXX-dependent interaction of F_c38-63 with caveolin 1 is not unique to this membrane-spanning protein. Our studies have indicated that caveolin 1 also associates with the chicken AE1-4 anion exchanger, and mutation of leucine 50 in its Y₄₇VEL tetrapeptide disrupts its association with caveolin 1. Interestingly, the AE1-4L50A mutant, which primarily resides in the basolateral membrane of MDCK cells, fails to acquire its complex N-linked sugars. These data strongly suggest that the recycling of AE1-4 from the plasma membrane to the Golgi (Adair-Kirk et al., 1999) is dependent upon its association with caveolin 1.

Depletion of cholesterol by treatment of cells with methyl--cyclodextrin dramatically alters the highly organized clusters of cell surface caveolin 1 in certain cell types, such as adipocytes (Kanzaki and Pessin, 2002). However, in contrast to adipocytes, caveolin 1 continued to accumulate in highly organized clusters in the basal membrane of methyl--cyclodextrin-treated MDCK cells. These clusters overlapped the distribution of both F_c38-63 (Fig. 6A-C) and AE1-4 (data not shown). Remarkably, the majority of surface-associated caveolin 1 undergoes internalization with F_c38-63 and AE1-4 to a common endosomal compartment when cellular cholesterol is repleted by treatment of cells with soluble cholesterol. Other investigators have shown that the relatively immobile cell surface population of caveolin 1 (Thomsen et al., 2002) can be induced to move following treatment of cells with glycosphingolipids or cholesterol (Sharma et al., 2004). Our results further demonstrate that the cholesterol content of the plasma membrane is a crucial determinant for regulating the mobility and/or internalization of the surface population of caveolin 1 and its associated cargo.

Recent studies have characterized a pathway for the endocytosis of Ctx-B that was dependent upon flotillin 1 (Glebov et al., 2006). This raft-associated protein colocalizes with Ctx-B on the cell surface and in early endosomes, and siRNA directed against flotillin 1 inhibited Ctx-B endocytosis (Glebov et al., 2006). However, the flotillin-associated endocytic pathway is dynamin-independent (Glebov et al., 2006), which distinguishes it from the dynamin-dependent endocytic pathway we have characterized for F_c38-63 in MDCK cells.

Previous investigators suggested that caveolin 1 plays a negative rather than a positive role in regulating caveolar-dependent endocytosis (Le et al., 2002). Our siRNA and mutagenesis studies are consistent with a positive regulatory role for caveolin 1 in directing F_c38-63 endocytosis in MDCK cells. Additional studies have shown that overexpression of caveolin 1 can both positively (Shigematsu et al., 2002) and negatively (Sharma et al., 2004; Kirkham et al., 2005) regulate the endocytosis of cargo. It is difficult to reconcile these various results with a single caveolin 1-dependent endocytic pathway. However, it is possible that the recruitment and internalization of different types of cargo by caveolin 1-dependent pathways can be differentially regulated. This differential regulation could in part be dependent upon whether cargos directly interact with caveolin 1 or simply reside in membrane microdomains enriched in caveolin 1.

	Materials and Methods

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Construction of siRNA and epitope-tagged constructs
A V5 epitope tag was introduced between amino acids 49 and 50 in the extracellular domain of F_c 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 extracellular loop of AE1-4. The tag was inserted between amino acids 545 and 546 of AE1-4 at an AgeI restriction site introduced by site-directed mutagenesis.

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

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

Transferrin internalization assays
MDCK cells were infected with adenovirus expressing the human transferrin receptor. 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-eight hours post-transfection, the cells were incubated with human transferrin conjugated to Alexa Fluor-594 for 30 minutes at 4°C in serum-free Dulbecco's modified Eagle's medium (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) containing 4% paraformaldehyde.

Cholesterol depletion and repletion
MDCK cells expressing Fc38-63 were washed with serum-free DMEM and incubated in serum-free DMEM containing 10 mM methyl--cyclodextrin for 1 hour 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 with 4% paraformaldehyde in PBS, and permeabilized by incubation in PBS containing 0.5% (vol/vol) Triton X-100 (PBST). The cells were then incubated with the rat 2.4G2 anti-F_c receptor monoclonal antibody and affinity-purified rabbit anti-caveolin 1 (Affinity BioReagents) or rabbit anti-furin antibodies (Affinity BioReagents). After washing, cells were incubated with anti-rat IgG conjugated to Alexa 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 permeable supports were incubated in the absence or presence of 10 mM methyl--cyclodextrin for 30 minutes at 4°C. During the 4°C incubation, the basolateral surface 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 the absence or presence of 10 mM methyl--cyclodextrin/cholesterol. At this time, the cells were fixed or they were shifted to 40 mM citric acid, 100 mM KCl, 135 mM NaCl (citrate buffer), pH 1.5, for 5 minutes at 4°C to elute surface-bound antibodies prior to fixation. The cells were then permeabilized and incubated with a rabbit caveolin-1 antibody, followed by anti-mouse IgG conjugated to Alexa Fluor-594 and anti-rabbit IgG conjugated to Alexa Fluor-488.

Cell surface binding and internalization assays
MDCK cells expressing F_c38-63 were grown on permeable supports. The basolateral surface of the cells was incubated with the rat anti-F_c 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 and incubated for 2 or 5 minutes at 37°C. The cells were then incubated in citrate buffer for 5 minutes at 4°C. Quantification of control experiments revealed that treatment of cells with citrate buffer prior to shifting the cells to 37°C elutes >99.9% of surface-bound antibodies (data not shown). Following the citrate wash, cells were rinsed in PBS, fixed and permeabilized with PBST. The cells were then incubated with rabbit antibodies against caveolin 1, clathrin (Sigma) or early endosomal antigen (EEA1, Affinity BioReagents) followed by the appropriate secondary antibodies. Z-stacks were collected through the cells using a Zeiss LSM510 confocal microscope. The percentage of pixels resulting from F_c38-63 staining that colocalized with the various markers in each slice in the z-stack was determined using the colocalization function in the Zeiss software.

In some instances, cells were simultaneously incubated with the rat anti-F_c 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 5 minutes 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. The percentage of pixels resulting from F_c38-63 staining in each slice of a z-stack that colocalized with transferrin or Ctx-B was determined.

In some experiments, F_c38-63-expressing cells were transfected with wild-type dynamin 2, dominant-negative dynamin 2 (K44A) (Altschuler et al., 1998) or with dominant-negative Eps1595-295. The dynamin cDNAs were cloned in 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-F_c receptor antibody for 30 minutes at 4°C. Following washing, the cells were incubated at 37°C for 30 minutes. The cells were then fixed and processed as described above.

F_c38-63-expressing cells were also transfected with the caveolin 1 or EGFP shRNA-expressing plasmids. Forty-eight hours post-transfection, the cells were incubated with the anti-F_c receptor antibody for 30 minutes at 4°C. Following washing, the cells were shifted to 37°C for 2 minutes. At this time, the cells were incubated 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 these constructs on F_c38-63 endocytosis was determined by comparing the total number of pixels in transfected cells with those present in neighboring cells that were non-transfected. Similar analyses investigated the effect of these reagents on transferrin and Ctx-B endocytosis.

Immunoprecipitation and immunoblotting
MDCK cells expressing V5-tagged versions of F_c38-63, F_c38-63Y47A, F_c38-63L50A, AE1-4 or AE1-4L50A were lysed in isotonic buffer containing 1% (vol/vol) Triton X-100 and processed for immunoprecipitation as described previously (Adair-Kirk et al., 1999) using anti-V5 antibodies. In some instances, precipitates were incubated with a glycosidase mixture (Calbiochem) that removes both O-linked and N-linked sugars. Alternatively, MDCK cells expressing untagged and V5-tagged versions of F_c38-63 were incubated with anti-V5 antibodies for 30 minutes at 4°C. The cells were then washed and lysed as described above, and immune complexes were isolated using protein A agarose. Immunoprecipitates prepared in both ways were subjected to immunoblotting analysis using the monoclonal anti-V5 antibody.

Lipid raft assays
MDCK cells expressing V5-tagged versions of F_c38-63, F_c38-63Y47A, F_c38-63L50A, AE1-4 or AE1-4L50A were rinsed with PBS and lysed in 2 ml of 20 mM Tris-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% sucrose in the same buffer lacking LubrolWX, and overlaid with a discontinuous sucrose gradient (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 were collected 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.

	Acknowledgments

 
This research was supported by a grant from the Southeast Affiliate of the American Heart Association (0355333B). Dynamin cDNAs were provided by D. Vignali. Adenoviral vectors expressing the transferrin 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 evaluation of the manuscript.

	Footnotes

 
^* Present address: Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USA

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