The recycling endosome of Madin-Darby canine kidney cells is a mildly acidic compartment rich in raft components

Abstract

We present a biochemical and morphological characterization of recycling endosomes containing the transferrin receptor in the epithelial Madin-Darby canine kidney cell line. We find that recycling endosomes are enriched in molecules known to regulate transferrin recycling but lack proteins involved in early endosome membrane dynamics, indicating that recycling endosomes are distinct from conventional early endosomes. We also find that recycling endosomes are less acidic than early endosomes because they lack a functional vacuolar ATPase. Furthermore, we show that recycling endosomes can be reached by apically internalized tracers, confirming that the apical endocytic pathway intersects the transferrin pathway. Strikingly, recycling endosomes are enriched in the raft lipids sphingomyelin and cholesterol as well as in the raft-associated proteins caveolin-1 and flotillin-1. These observations may suggest that a lipid-based sorting mechanism operates along the Madin-Darby canine kidney recycling pathway, contributing to the maintenance of cell polarity. Altogether, our data indicate that recycling endosomes and early endosomes differ functionally and biochemically and thus that different molecular mechanisms regulate protein sorting and membrane traffic at each step of the receptor recycling pathway.

Figure 1

Characterization of the hTfR-expressing MDCK cell lines. (A) Total cell extracts prepared from untransfected MDCK cells and cells expressing hTfR or m-hTfR were analyzed by SDS-PAGE and Western blotting with the use of antibodies against hTfR. (B) Cell surface distribution of TfR was measured by incubating the apical (ap) or basolateral (bl) side of filter-grown hTfR or m-hTfR cells with Tf-HRP at 4°C. Results are presented as percentages of total cell-associated HRP activity. (C) Continuous Tf-HRP uptake at 37°C was measured in polarized m-hTfR cells as a function of time. Cells were incubated with Tf-HRP. At the indicated times, cell-associated HRP activity was quantified and normalized to total cellular protein. (D) TfR (red) topology in polarized m-hTfR cells was analyzed by immunofluorescence confocal microscopy with the use of antibodies against hTfR. Tight junctions (green) were identified with the use of antibodies against ZO1. The distance between the section plane and the filter is indicated on each panel. (E) Cellular distribution of endocytosed Tf–rhodamine. Tf–rhodamine was bound to the basolateral plasma membrane at 4°C and internalized at 37°C for the indicated times. Confocal microscope sections taken through the apical (ap), lateral (lat), and basal (bas) regions of the cells are shown.

Figure 2

Purification of TfR-positive endosomes. (A) Lower panel, PNSs prepared from cells expressing m-hTfR were fractionated on a discontinuous sucrose gradient loaded with 8, 20, and 35% sucrose. After centrifugation, fractions were collected and analyzed by SDS-PAGE and Western blotting with the use of anti-TfR antibodies as in Figure 1A. Lanes were loaded with either the same amount of protein (enrichment) or 10% of the volume (yield) of each fraction. Upper panel, To label the plasma membrane (PM), Tf-HRP was bound to the surface of m-hTfR cells at 4°C. To label recycling endosomes (TfE), Tf-HRP was bound to the surface of m-hTfR cells at 4°C and internalized for 10 min at 37°C. PNSs were prepared and fractionated, and the HRP content of the fractions was quantified. (B) Membranes enriched in m-hTfR were prepared with the use of the sucrose gradient, as in A, and further fractionated by immunoisolation with the 9E10 mAb against the myc tag and magnetic beads. As a control for nonspecific binding, the whole purification procedure was carried out in parallel with hTfR cells. Beads were retrieved with a magnet, and then both bound material (B) and unbound material sedimented at 150,000 × g for 30 min (UB) were analyzed for TfR content by Western blotting as in Figure 1A. (C) The purification protocol was carried out as in B with the use of m-hTfR and hTfR cells that had been metabolically labeled with [³⁵S]Met, and samples were analyzed by high-resolution two-dimensional gel electrophoresis followed by autoradiography. The general protein pattern of the immunoisolated fraction [Bound m-hTfR (specific)] was compared with that of the unbound material sedimented at 150,000 × g for 30 min [Unbound m-hTfR] to determine which proteins were specifically immunoisolated (examples are indicated with red arrowheads). The asterisk indicates the position of flotillin-1 in the same gel system. Examples of proteins that were not isolated are indicated with black arrows. Contaminants (blue arrowheads) were determined by comparison with samples from hTfR cells [Bound hTfR (unspecific)]. The position of actin, which is commonly used for orientation in this type of analysis, is indicated on the unbound m-hTfR gel.

Figure 3

Protein composition of TfR-positive endosomes. Endosomes containing m-hTfR were purified, with the use of cells expressing hTfR as a control, as in Figure 2, B and C. (A and B) Immunoisolated membranes bound to the beads (B) were analyzed by SDS-PAGE and Western blotting with the use of specific antibodies against the indicated proteins. For comparison, 50% of the total fraction used as starting material for immunoisolation (IN) was also analyzed. Asterisks mark the light chain of the antibody used for immunoisolation. (C) Immunoisolated membranes (B) are compared with unbound material sedimented at 150,000 × g for 30 min (UB) as in Figure 2B. Analysis was as in A and B, with the use of specific antibodies against the indicated proteins.

Figure 4

Recycling endosomes in MDCK cells are less acidic than early endosomes. Tf-FITC was prebound at 4°C to m-hTfR cells and then internalized for 5 min at 20°C (upper panels) or internalized continuously for 20 min at 37°C (lower panels). Typical examples of the respective labels are shown. Because of technical constraints, we used subconfluent cells grown on glass coverslips rather than on filters. The pH of individual organelles was measured by fluorescence ratio imaging of internalized Tf-FITC. The histograms show the pH distribution of 767 and 206 endosomes for the upper and lower panels, respectively, with the mean ± SD given in the left panels.

Figure 5

The apical endocytic pathway intersects the Tf recycling pathway. (A) m-hTfR cells were allowed to endocytose Tf–rhodamine from the basolateral medium for 30 min and dextran–Oregon Green (dextran–OG) from the apical medium for the last 10 min, and cells were analyzed by confocal fluorescence microscopy. Confocal sections in the apical (Ap) or basolateral (Bl) region of the cell are shown. Large arrows indicate organelles positive for both Tf–rhodamine and dextran–OG. Small arrows indicate organelles that contain only dextran–OG, and arrowheads indicate organelles that contain only Tf–rhodamine. (B) Cells expressing m-hTfR were allowed to endocytose HRP from the apical medium for 10 min (pulse) or for 15 min followed by a 30-min incubation in marker-free medium (chase). Endosomes containing m-hTfR were immunoisolated as in Figure 2, B and C. Bound material and unbound material sedimented at 150,000 × g for 30 min were analyzed for HRP activity. Results are expressed as percentages of the total HRP activity.

Figure 5

The apical endocytic pathway intersects the Tf recycling pathway. (A) m-hTfR cells were allowed to endocytose Tf–rhodamine from the basolateral medium for 30 min and dextran–Oregon Green (dextran–OG) from the apical medium for the last 10 min, and cells were analyzed by confocal fluorescence microscopy. Confocal sections in the apical (Ap) or basolateral (Bl) region of the cell are shown. Large arrows indicate organelles positive for both Tf–rhodamine and dextran–OG. Small arrows indicate organelles that contain only dextran–OG, and arrowheads indicate organelles that contain only Tf–rhodamine. (B) Cells expressing m-hTfR were allowed to endocytose HRP from the apical medium for 10 min (pulse) or for 15 min followed by a 30-min incubation in marker-free medium (chase). Endosomes containing m-hTfR were immunoisolated as in Figure 2, B and C. Bound material and unbound material sedimented at 150,000 × g for 30 min were analyzed for HRP activity. Results are expressed as percentages of the total HRP activity.

Figure 6

Raft components are enriched in purified recycling endosomes. Endosomes containing m-hTfR were purified, with the use of cells expressing hTfR as a control, as in Figure 2, B and C, from cells that had been metabolically labeled with [³²P]orthophosphate (for phospholipids) or [¹⁴C]acetate (for cholesterol). Lipids were extracted and analyzed by two-dimensional TLC (phospholipids) or one-dimensional TLC (cholesterol). (A) Two-dimensional TLC of ³²P-labeled immunoisolated fractions from cells expressing m-hTfR (Bound) compared with the starting material (IN). The same amounts (dpm) were loaded in each case. Only samples from m-hTfR cells are shown in this typical experiment. (B) Radioactivity was quantified with the PhosphorImager. Indicated values correspond to the enrichment over the starting material (expressed as relative specific activity), normalized to nonspecific binding measured with the use of hTfR cells. SM, sphingomyelin; PI, phosphatidylinositol; PS, phosphatidylserine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; chol., cholesterol.

Figure 7

Raft components distribute to recycling endosomes. (A) Filter-grown m-hTfR cells were allowed to incorporate sphingomyelin (SM)–BODIPY for 30 min from both sides of the filters. Simultaneously, Tf–rhodamine was internalized from the basolateral side of the filters. Confocal sections in the apical (Ap) or basolateral (Bl) region of live cells are shown. Arrowheads indicate intracellular colocalization between sphingomyelin–BODIPY and Tf–rhodamine in both the apical and basolateral regions of the cells. (B) Cells expressing m-hTfR grown on coverslips were processed for double-label immunofluorescence analysis, with the use of antibodies against hTfR as in Figure 1C, and filipin to reveal the distribution of cholesterol. Arrowheads indicate intracellular colocalization between cholesterol and TfR.

Figure 8

Caveolin copurifies with the Tf receptor. (A) Cells expressing m-hTfR were fractionated on a discontinuous sucrose gradient loaded with 8, 20, and 35% sucrose, as in Figure 2A. Fractions were collected and analyzed by SDS-PAGE and Western blotting with the use of antibodies against caveolin or TfR as a control. Lanes were loaded either with the same amount of protein (enrichment) or with 10% of the volume (yield) of each fraction. As controls, the figure also shows the distribution of annexin II and Arf6, which were not detected in the immunoisolated fractions. (B) Amounts of caveolin and TfR were quantified by scanning the gels in a typical experiment; yields [%] and enrichment [×] are indicated, both for the 20/30% gradient interface (as in A) and for the immunoisolated fractions (as in Figure 3A).

Figure 9

Immunoelectron microscopic localization of caveolin-1 in MDCK cells. Filter-grown MDCK cells were perforated with the use of nitrocellulose to remove parts of the apical surface. Cells were labeled for caveolin-1, fixed, and processed for Epon embedding. (A) Image shows an area of the lateral membrane with gold labeling associated with caveolae (arrowheads). (B–D) Images show areas underlying the apical plasma membrane. Labeling for caveolin-1 is associated with extensive tubular structures (arrows). Bars, 100 nm.