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First published online 23 May 2006
doi: 10.1242/jcs.02978

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Flat clathrin coats on endosomes mediate degradative protein sorting by scaffolding Hrs in dynamic microdomains

Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital and The University of Oslo, Montebello, N-0310 Oslo, Norway

View larger version (29K): [in a new window]   Fig. 1. Interaction of Hrs with clathrin. (A) Schematic representation of the mouse Hrs protein, with its different domains indicated. Proline- and proline/glutamine-rich regions are also indicated. VHS, domain conserved in Vps27, Hrs and STAM; FYVE, domain conserved in Fab1, YOTB, Vac1 and EEA1; UIM, uibiquitin-interacting motif; CC, coiled-coil domain; CB, clathrin-box motif (the residues are indicated in single-letter code). (B) Recombinant GST-clathrin-TD was immobilised on glutathione-Sepharose beads and incubated with cell lysate from HeLa cells transiently transfected with the Hrs constructs indicated. The beads were pelleted and analysed by SDS-PAGE and immunoblotting with an anti-myc antibody. (C) Recombinant GST or GST-clathrin-TD were immobilised on glutathione-Sepharose beads and incubated with cell lysate from HeLa cells transiently transfected with Hrs. The cells were serum-starved for 4 hours and stimulated or not with 50 ng/ml EGF for 15 minutes before lysis. The beads were pelleted and analysed by SDS-PAGE and immunoblotted with an anti-myc antibody. The lysate was also subjected to immunoprecipitation with the anti-myc antibody and immunoblotted with an anti-phosphorylated-tyrosine antibody to detect phosphorylated Hrs. (D) HEp-2 cells were transfected with Hrs-specific siRNA or a scrambled-sequence RNA and subjected to western blotting to detect the efficiency of Hrs knockdown (upper panel). Tubulin is shown as a loading control (lower panel). CB, clathrin-box; IB, immunoblot, IP, immunoprecipitation; TD, terminal domain; PS, Ponceau S-staining.

View larger version (72K): [in a new window]   Fig. 2. Hrs and its clathrin-box motif are important for recruitment of clathrin to early endosomes. (A,B) Cells treated with a (A) scrambled control RNA duplex or (B,C) siRNA against Hrs were prepared for immunofluorescence microscopy and labelled with antibodies against clathrin, EEA1 and Hrs. Clathrin was easily detected on EEA1- and Hrs-positive endosomes in control cells (A, inset). In Hrs-depleted cells (arrows), the endosomes were devoid of clathrin (B,C, inset with dashed lines). The siRNA-treated cells were transiently transfected with the siRNA-resistant Hrs constructs indicated, to reintroduce Hrs. Arrowheads point at siRNA-treated cells re-transfected with myc-Hrswt (B) or myc-HrsCB (C). In siRNA-treated cells transfected with myc-Hrswt, endosomal clathrin was detected (B, inset with solid lines). In siRNA-treated cells transfected with myc-HrsCB, endosomes were virtually devoid of clathrin (C, inset with solid lines). White in merged images indicates colocalisation of Hrs, EEA1 and clathrin. Bar, 10 µm.

View larger version (47K): [in a new window]   Fig. 3. Endosomal clathrin concentrates Hrs in EEA1-negative microdomains. (A,B) HeLa-cells were co-transfected with plasmid-based clathrin-specific siRNA and Rab5Q79L, and stained with anti-clathrin, anti-Hrs or anti-EEA1 antibodies for immunofluorescence confocal microscopy. (A) Control cell with endosomal clathrin. (B) siRNA-transfected cell where clathrin is efficiently knocked down. Yellow indicates colocalisation of Hrs and clathrin, and cyan indicates colocalisation of Hrs and EEA1 in the merged images. Insets show enlarged endosomes. The amount of endosomal Hrs that colocalise with EEA1 was quantified as described in Materials and Methods (C). Error bars give the mean ± s.e.m. Control, n=25; siRNA clathrin, n=27. (D,E) HEp-2 cells were transfected with Hrs-specific siRNA oligonucleotides. The siRNA-treated cells were transiently co-transfected with Rab5Q79L and myc-Hrswt (D) or myc-HrsCB (E). Insets show two different enlarged endosomes from each transfection. Yellow indicates colocalisation of Hrs and EEA1. Bars, 10 µm. (F) The amounts of endosomal myc-Hrswt or myc-HrsCB that colocalised with EEA1 were quantified. Error bars give the mean ± s.e.m. Myc-Hrswt, n=14; myc-HrsCB, n=14.

View larger version (29K): [in a new window]   Fig. 4. A PtdIns(3)P probe is redirected from EEA1-positive regions into Hrs-containing microdomains through clathrin binding. (A) Schematic representation of the 2xFYVE-Hrs CT chimeric constructs, with and without the clathrin-box motif. HeLa cells were transiently co-transfected with Rab5Q79L and (B) myc-2xFYVE-Hrs CT or (D) myc-2xFYVE-Hrs CTCB, and stained with anti-myc and anti-EEA1 antibodies for immunofluorescence confocal microscopy. Alternatively, cells were triple transfected with Rab5Q79L and (C) myc-2xFYVE-Hrs CT and YFP-Hrs or (E) myc-2xFYVE-Hrs CTCB and YFP-Hrs, and stained with anti-myc antibodies. Insets show two different enlarged endosomes from each transfection. Yellow indicates colocalisation. Bar, 5 µm. The level of colocalisation of the different proteins on endosome membranes was quantified as described in Materials and Methods (F). Error bars give the mean + s.e.m. myc-2xFYVE-Hrs CT + EEA1, n=19; myc-2xFYVE-Hrs CT + YFP-Hrs, n=27; myc-2xFYVE-Hrs CTCB + EEA1, n=27; 2xFYVE-Hrs CTCB + YFP-Hrs, n=29.

View larger version (51K): [in a new window]   Fig. 5. Membrane-associated Hrs and clathrin are rapidly exchanged with cytosolic pools. HeLa cells were co-transfected with Rab5Q79L and GFP-clathrin LC or YFP-Hrs, and studied live at 25°C as described in Materials and Methods. After photobleaching of whole endosomes, both Hrs and clathrin recovered in microdomains on the limiting membrane of the enlarged endosomes. (A) Representative images of GFP-clathrin LC and YFP-Hrs. (B) Representative recovery curve for YFP-Hrs. (C) Representative recovery curve for GFP-clathrin LC. The half time of the recovery, t1/2, was calculated from several recordings. YFP-Hrs, n=12; GFP-clathrin LC, n=9.

View larger version (39K): [in a new window]   Fig. 6. Tsg101 is found in Hrs-containing microdomains. HEp-2 cells were transfected with Rab5Q79L and stained for immunofluorescence confocal microscopy with anti-Tsg101 and anti-EEA1 or anti-Hrs antibodies. (A) Two selected enlarged endosomes. Yellow indicates colocalisation. (B) Level of colocalisation of Tsg101 and EEA1 or Hrs on membranes of Rab5Q79L-containing endosomes, quantified as described in Materials and Methods. Error bars give the mean + s.e.m. EEA1, n=16; Hrs, n=19, P=0.008.

View larger version (25K): [in a new window]   Fig. 7. Clathrin binding by Hrs is important for EGF degradation. (A,B) For EGF degradation assay, HEp-2 cells were treated with Hrs-specific siRNA oligonucleotides and at the same time transiently transfected with (A) siRNA-resistant myc-Hrswt or (B) myc-HrsCB. The cells were stimulated with 100 ng/ml Rhodamine-labelled EGF for 15 minutes, washed and incubated for 3 hours to allow degradation of internalised EGF. The cells were then prepared for confocal immunofluorescence microscopy and labelled with anti-Hrs and anti-myc (not shown) antibodies. (A) Hrs-depleted cells transfected with myc-Hrswt showed normal degradation of EGF (arrowheads) compared with Hrs-depleted neighbouring cells, where the degradation was inhibited. (B) Hrs-depleted cells transfected with myc-HrsCB accumulated EGF (arrowheads) similar to Hrs-depleted neighbouring cells. White lines indicate the cell borders. Bar, 10 µm. Arrows point at endosomes where myc-HrsCB and EGF colocalise. (C) The amount of EGF remaining after 3 hours of chase was quantified as described in Materials and Methods and represented as percentage of total amount of internalised EGF after 15 minutes. Control cells were treated with scrambled RNA. Error bars give the mean + s.e.m. from four independent experiments. Control, n=33 cells; siRNA, n=96; siRNA + myc-Hrswt, n=73; siRNA + myc-HrsCB, n=89.

View larger version (18K): [in a new window]   Fig. 8. Model of endosomal protein sorting. (i) Ubiquitylated cargo is retained in a Hrs/clathrin microdomain on the endosome membrane. (ii) The exchange of Hrs and clathrin with a cytosolic pool generates space for the recruitment of ESCRT-I into the coat. (iii) Cargo is efficiently transferred from Hrs to ESCRT-I within the coat, which is maintained through the presence of clathrin. (iv) As the endosome matures, Hrs and clathrin dissociate from the endosome and ESCRT-I remains bound to cargo on the endosome membrane. ESCRT-II and ESCRT-III are recruited, which promotes further sorting of cargo into the forming MVE.