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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

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.

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.

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.

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.

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.

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