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First published online 16 October 2007
doi: 10.1242/jcs.003343

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Clathrin-mediated endocytosis of a lipid-raft-associated protein is mediated through a dual tyrosine motif

¹ Department of Biochemistry, University of Bristol, Bristol, BS8 1T, UK
² Zentrum für Biochemie und Molekulare Zellbiologie, Universität Goettingen, Germany

View larger version (36K): [in this window] [in a new window]   Fig. 1. CD317 is internalised through a clathrin-dependant pathway. (A) H4IIE cells transiently expressing CD317-GFP were imaged and the highlighted area was photo-bleached and allowed to recover for 17 minutes. Images were taken every minute, Cells were incubated in the presence of 200 µg/ml cycloheximide for 1 hour prior to imaging (and in the continued presence of cycloheximide during imaging). Bar, 10 µm. (B) Transiently transfected H4IIE cells expressing dominant-negative AP180-C-myc were subjected to uptake of transferrin (Tfr), cholera-toxin-B subunit (CT-B) or CD317 antibody uptake for 15 minutes: cells were then acid washed to remove any non-internalised material. Asterisks denote transfected cells. Bars, 10 µm. (C) Immunoblot of fractions obtained during the isolation of purified clathrin-coated vesicles (CCVs) from rat brain, which had been separated by SDS-PAGE (12% gel), using anti-CD317 antibody (upper panel), or anti-GSK3 antibody (lower panel). Lane 1, purified CCVs; lane 2, peak 1 from the purification of CCVs (containing vesicles depleted in CCVs); lane 3, cytosol; lane 4, post-nuclear fraction; lane 5, total rat brain lysate. The same blot was initially probed with anti-GSK3 antibody, stripped and re-probed with anti-CD317 antibody. The fractions are the same as those previously described and characterised (Korolchuk and Banting, 2002). (D) HeLA cells transiently transfected with a construct encoding wild-type CD317. After 24-hour-expression, cells were subjected to antibody uptake (using a CD317 antibody) and Tfr uptake for 2 minutes, or just antibody uptake for 6 minutes. Images show colocalisation between EEA1 or Tfr and CD317 as indicated. Indicated areas have been enlarged. Colocalisation between EEA1 and CD317 is 64.1% (n=310) and between Tfr and CD317 40% (n=233). Bars, 10 µm.

View larger version (33K): [in this window] [in a new window]   Fig. 2. The role of the cytosolic domain in the internalisation of CD317. (A,B) COS cells were transiently transfected with constructs encoding either full-length, truncated or mutated CD317 as indicated and subjected to anti-CD317 antibody uptake for 15 minutes. CD317-antibody uptake is clearly inhibited in cells expressing either truncated CD317 (MDER panel) or CD317 with both Y6A and Y8A mutations (MAPSFAHA panel), as shown by the accumulation of antibody at the plasma membrane in these cells. By contrast, the punctate intracellular fluorescent signal shows that there is efficient internalization of CD317 in cells expressing either the Y6A (MAPSFAHY) or Y8A (MAPSFYHA) mutations. The cytosolic N-terminal sequence of full-length rat CD317 is MAPSFYHYLPVAMDER. The MDER construct lacks the N-terminal 12 amino acids of the cytosolic domain, the MAPSFAHY construct has a Y6A mutation, the MAPSFYHA construct has a Y8A mutation and the MAPSFAHA construct has the point mutations Y6A and Y8A. All mutations are in the context of the full-length cytosolic domain. Bar, 10 µm. ID, intracellular domain; TM, transmembrane domain; ED, extracellular domain and GPI; glycosylphosphatidylinositol anchor.

View larger version (35K): [in this window] [in a new window]   Fig. 3. The cytosolic domain of CD317 binds the AP1 and AP2 adaptor-complex subunits µ1 and µ2. (A) Immunoblots, probed with an anti-His tag antibody, of pull-down assays between a biotinylated N-terminal peptide of CD317 and His-tagged thioredoxin (His-TRX)-fusion proteins of the µ subunits of the AP1 and AP2 adaptor complexes or LDH. Lanes are labelled according to the loaded lysate (10% of input for pull-down); biotin-labelled lanes represent material isolated using biotin-coated beads and CD317-labelled lanes material isolated using beads coated with biotinylated CD317 peptide, pep1 and pep2 refer to specific and non-specific competing peptides. Markers indicate molecular mass in kDa. (B) Immunoblots of lysates from HeLa cells that had been transfected with siRNA targeting µ2 (µ2 siRNA) or siRNA targeting lamin A/C (lamin A/C siRNA) and probed with anti-µ2 or anti-lamin antibodies as indicated. Blots were then stripped and reprobed with an anti-tubulin antibody as a loading control. (C) HeLa cells that had been transfected with siRNA targeting µ2 or siRNA targeting lamin A/C (as indicated) were used for uptake of transferrin, EGF or CD317 antibody for 15 minutes and then acid-washed to remove any non-internalised material. Bars, 10 µm. (D) Quantification of the data presented in C. PI/cell, pixel intensity per cell. Transferrin uptake in µ2-knockdown cells is 8% of control (n=106 and 168), CD317 uptake in µ2-knockdown cells is 15.2% of control (n=20 and 21), EGF uptake is not affected (n=118 and 92).

View larger version (34K): [in this window] [in a new window]   Fig. 4. CD317 is mislocalised in µ1A-deficient cells. CD317 was transiently expressed in µ1A-deficient cells (µ1–/–) and in control cells (Clone24) for 24 hours then antibody uptake was undertaken for 30 minutes. Cells were then processed for immunofluorescence analysis using antibodies against either GM130 or M6PR, or processed for fluorescence analysis using Lysotracker. Colocalisation between CD317 and M6PR is 43.9% n=129. Bars, 10 µm.

View larger version (29K): [in this window] [in a new window]   Fig. 5. Removal of the GPI anchor impedes CD317 internalisation. (A) H4IIE cells were incubated for 1 hour at 37°C in serum-free medium with or without PI-PLC as indicated, and subjected to anti-CD317 antibody uptake for 20 minutes at 37°C prior to fixation and processing for immunofluorescence analysis. Bars, 10 µm. (B) Immunoblot analysis of fractions from sucrose-density-gradient separation of H4IIE cell lysates from control cells (Control) and from cells that had been incubated in PI-PLC prior to lysis (PIPLC). Fractions were taken from the top of the gradient (i.e. fraction 1 is the most buoyant), and blots were probed with CD317 antibody. (C) Surface proteins of HeLa cells were treated with PI-PLC or serum-free medium and then biotinylated. Endocytosis was allowed to proceed for 0, 2, 4 or 8 minutes after which surface biotin was removed. Internalised proteins were pulled down with streptavidin beads and analysed by western blotting with antibodies against transferrin receptor and CD317.