QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 18 January 2005
doi: 10.1242/jcs.01628

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Seet, L.-F. Articles by Hong, W. Search for Related Content PubMed PubMed Citation Articles by Seet, L.-F. Articles by Hong, W. Social Bookmarking                         What's this?

Endofin recruits clathrin to early endosomes via TOM1

Membrane Biology Laboratory, Institute of Molecular and Cell Biology, Proteos, 61 Biopolis Drive, Singapore 138673, Republic of Singapore

View larger version (48K): [in a new window]   Fig. 1. Interaction between TOM1 and clathrin. (A) Identification of clathrin as a binding protein for TOM1. 300 µg recombinant GST or GST-TOM1(300-492) immobilized on glutathione-Sepharose beads, were incubated overnight with 85 mg pre-cleared rat brain cytosol at 4°C. Upon washing, proteins bound to the columns were eluted and resolved by SDS-PAGE and visualized by Coomassie Blue staining of the gel. Peptide sequences derived from the 220 kDa protein band were obtained by mass spectrometry: 1. AYEFAER; 2. TPDTIRR; 3. HELIEFR; 4. ENPYYDSR. (B) The C-terminal region of TOM1 interacts with clathrin. GST or GST-TOM1(300-492) immobilized on glutathione-Sepharose beads were incubated with A431 cytosol. The beads were washed and bound proteins as well as input (4%) were resolved by SDS-PAGE. 50 µg of input lysate and post-pull-down extracts were precipitated with trichloroacetic acid and analyzed by SDS-PAGE. Immunoblot analysis was performed with anti-clathrin heavy chain (HC) antibodies (upper and middle panels). GST and GST fusions were visualized by immunoblotting with anti-GST antibody as a control for levels of fusions used in pull-down experiments (bottom panel).

View larger version (34K): [in a new window]   Fig. 2. Optimal interaction of TOM1 with clathrin requires residues 300-321 and 362-366. (A) Schematic diagram of TOM1 deletion constructs used in the clathrin pull-down experiments. VHS, VPS-27, Hrs and STAM; GAT, GGAs and TOM1 homology. (B) Residues 300-366 are required to interact with clathrin. Glutathione-sepharose-bound recombinant GST fusion proteins of the deletion constructs depicted in A were incubated with A431 cytosol and proteins bound to the beads and input were resolved by SDS-PAGE analysis followed by western blotting with anti-clathrin heavy chain antibodies. GST and GST fusions from the same gel were visualized by Coomassie Blue staining as a control for levels of fusions used in the pull-down experiments. 50 µg input cytosol and proteins from post-pull-down extracts were precipitated with TCA and similarly analyzed by SDS-PAGE and immunoblotting with anti-clathrin heavy chain and anti-ß-tubulin antibodies, the latter to indicate loading levels.

View larger version (31K): [in a new window]   Fig. 3. Mutation of the putative clathrin-binding box at amino acids 362-366 partially disrupts TOM1 association with clathrin. (A) Schematic diagram of TOM1 deletion constructs with or without mutation of 362Leu and 364Asp to Ala. (B) Mutation of 362LEDEF366 reduces the ability of TOM1 to pull down clathrin. Glutathione-sepharose-bound recombinant GST fusion proteins of the constructs depicted in A were incubated with A431 cytosol and analyzed as in Fig. 2B.

View larger version (32K): [in a new window]   Fig. 4. Optimal interaction of TOM1 with clathrin involves three sites: residues 300-321, 321-326 and 362-366. (A) Schematic diagram of TOM1 C-terminal deletion constructs used in the clathrin pull-down experiments. The relative efficiency with which each construct pulled down clathrin as observed in B is indicated on the right as + or -. (B) The clathrin-binding domain of TOM1 includes one putative clathrin-binding box at 321-326, which is sufficient by itself whereas residues 300-321 and 362-366 enhance the clathrin-binding efficiency. The GST fusion constructs shown in A were used to pull down cytosolic clathrin and analyzed as described in Fig. 2B. The upper panels indicate the amounts of clathrin pulled down by 20 µg of GST or GST fusion proteins. Input represents 50 µg of the starting material. The bottom panels are Coomassie-stained versions of the same experiments. (C) Schematic diagram depicting the location of the sites in TOM1 that are critical for optimal clathrin interaction. CB, clathrin-binding site; CB-I and CB-II: clathrin-binding enhancing sites I and II, respectively; EBD, endofin-binding domain; GAT, GGAs and TOM1 homology; VHS, VPS-27, Hrs and STAM.

View larger version (97K): [in a new window]   Fig. 5. Overexpressed endofin recruits clathrin to early endosomes. A431 cells were transfected with Myc-tagged endofin and processed for immunofluorescence analysis by fixing followed by permeabilization with 0.05% saponin. The cells were stained with anti-Myc (left panels and green in merged images) and anti-clathrin heavy chain (A), anti-dynamin (B), anti-AP1 (C), anti-AP2 (D) or anti-AP3 (E) (middle panels and red in merged images on right) and analyzed by confocal microscopy. Areas of yellow coloration in merged images indicate colocalization. Bar, 10 µm.

View larger version (48K): [in a new window]   Fig. 6. The C-terminal region of endofin cannot be replaced by the corresponding region of SARA for interaction with the C-terminal domain of TOM1 and its membrane recruitment. (A) Schematic of wild-type endofin and SARA constructs as well as the chimeric constructs used in the pull-down experiments. (B) The C-terminal region of endofin but not SARA mediates binding to the TOM1 C-terminus. 293T cells were transfected with the Myc-tagged constructs depicted in A and the resultant lysates were incubated with either GST or GST-TOM1(300-492), which were bound to glutathione-Sepharose beads. The inputs (3%) as well as pull-down materials were resolved by SDS-PAGE and analyzed by western blotting with anti-Myc (upper panel) and anti-GST (lower panel) antibodies, the latter to indicate levels of GST or GST fusion proteins used in the experiment. (C) Membrane recruitment of TOM1 by endofin is dependent on the C-terminus of endofin, which cannot be replaced by the corresponding region of SARA. A431 cells were transfected with each of the Myc-tagged constructs depicted in A. Upon fixation and permeabilization, the cells were stained with antibodies against the Myc epitope (left panels) and endogenous TOM1 (right panels). (D) Membrane recruitment of clathrin by endofin is dependent on the C-terminus of endofin, which cannot be replaced by the corresponding region of SARA. A431 cells were transfected with the Myc-tagged constructs depicted in A. Upon fixation and permeabilization, the cells were stained with antibodies against the Myc epitope (left panels) and clathrin heavy chain (HC) (right panels). Bar, 10 µm.

View larger version (68K): [in a new window]   Fig. 7. The chimera HA-FYVE2-TOM1 behaves like a FYVE domain protein. (A) Schematic diagram of the chimeric construct HA-FYVE2-TOM1 where two FYVE domains derived from endofin were fused N-terminal to the full-length TOM1 coding sequence. HA-FYVE2-TOM1 causes aggregation/fusion of early endosomes but has no effect on the clathrin-associated adaptor proteins AP1, AP2 or AP3. A431 cells were transfected with HA-FYVE2-TOM1 and processed for immunofluorescence analysis by fixation with paraformaldehyde followed by permeabilization with either 0.1% Triton X-100 (B and C) or 0.05% saponin (D-F). The cells were stained with anti-HA (left panels and green in merged images) and anti-EEA1 (B), anti-endofin (C), anti-AP1 (D), anti-AP2 (E) or anti-AP3 (F) (middle panels and red in merged images on the right) and analyzed by confocal microscopy. Yellow in merged images represents areas of colocalization. CB, CB-I and CB-II, clathrin-binding sites; EBD, endofin-binding domain; GAT, GGAs and TOM1 homology; VHS, VPS-27, Hrs and STAM. Bar, 10 µm.

View larger version (54K): [in a new window]   Fig. 8. HA-FYVE2-TOM1 chimera recruits clathrin to endosomes while mutation of CB-I and CB-II reduces this activity. (A) Schematic diagram of the chimeric constructs used in the experiment. Mutated residues are indicated in italics and deletions are indicated by a gap. (B) HA-FYVE2-TOM1 recruits clathrin to endosomes whereas the chimeric CB-I and CB-II single or double mutants are significantly less effective. A431 cells were transfected with each of the constructs depicted in A and processed for immunofluorescence analysis by fixing with paraformaldehyde followed by permeabilization with 0.05% saponin. The cells were stained with anti-HA (left panels and green in merged images) and anti-clathrin heavy chain (middle panels and red in merged images) and analyzed by confocal microscopy. Merged images are shown in the right panels where yellow represents areas of colocalization. CB, CB-I and CB-II, clathrin-binding sites; EBD, endofin-binding domain; GAT, GGAs and TOM1 homology; VHS, VPS-27, Hrs and STAM. Bar, 10 µm.

View larger version (63K): [in a new window]   Fig. 9. TOM1-specific antibody can block the ability of the C-terminal region of TOM1 to bind clathrin. (A) Recombinant GST-TOM1(300-492) bound to glutathione-sepharose beads was pre-incubated with the indicated amounts of anti-TOM1 antibodies or rabbit IgG (as control). The beads were then washed and incubated with A431 cytosol in pull-down experiments. The input (10%) and pull-down materials were resolved by SDS-PAGE and analyzed by immunoblotting with either anti-clathrin heavy chain (upper panel) or anti-GST (lower panel) antibody. The amounts of TOM1 antibodies bound to the GST fusion beads were visualized by western blotting directly with anti-rabbit IgG conjugated to horseradish peroxidase (middle panel). (B) TOM1-specific antibody disrupts the cellular distribution of clathrin. A431 cells were microinjected with anti-TOM1 or rabbit IgG as a control. The cells were then fixed and permeabilized with 0.05% saponin before staining with FITC-conjugated anti-rabbit IgG to visualize the microinjected antibodies (left panels) and anti-clathrin heavy chain (right panels) to detect the distribution patterns of clathrin. Bar, 10 µm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter