PMC

Interactions observed within the FHF complex and between the FHF complex and the HOPS complex. See text for details.

FTS and Hook proteins associate with class B and class C components of the HOPS complex. (A–E) Plasmids expressing the indicated HA-tagged Vps or FLAG-tagged Hook constructs were transfected into HEK293T cells and after 48 h, protein complexes were purified using the indicated anti-HA or anti-FLAG (M2) antibodies. Washed complexes and lysates as loading controls were subjected to SDS-PAGE and immunoblotting. (F–H) Plasmids expressing the indicated HA-tagged Vps or GST-tagged FTS constructs were transfected into HEK293T cells and after 48 h, protein complexes were purified using GSH-Sepharose. Washed complexes and lysates as loading controls were subjected to SDS-PAGE and immunoblotting. (I) Association of endogenous Hook1 with endogenous Vps18. HeLa cells were lysed in either buffer containing NP-40 or buffer containing CHAPS (see Materials and Methods), and cleared extracts were subjected to immunoprecipitation by using anti-Hook1 antibodies. Immune complexes were immunoblotted with either anti-Vps18 or anti-Hook1 antibodies.

The FHF complex promotes timely trafficking of EGF through the endosomal pathway. (A) Fluorescently labeled EGF (EGF-TR, Texas Red) was used in endosomal trafficking assays as described in Materials and Methods. At either 5 min or 2 h after initiation of EGF trafficking, cells were fixed and stained with antibodies against EEA1, CD63, or LAMP1 by using anti-FITC detection (green). Cells were imaged by confocal microscopy. (B) The extent of overlap between the indicated endosomal markers and EGF-TR was determined using MetaMorph software as described in Materials and Methods. For each condition, 30–50 cells were imaged from three independent experiments. The percentage of EGF-TR that colocalizes with marked endosomes.

p107^FHIP interacts with FTS and Hook proteins to form the FHF complex. (A) GST-p107^FHIP interacts with FTS and Hook1. Plasmids expressing the indicated FLAG-tagged wild-type or mutant FTS proteins were transfected into HEK293T cells together with plasmids expressing either GST-p107^FHIP or GST and after 48 h, cell extracts were generated and incubated with GSH-Sepharose. Bound proteins and control lysates were subjected to immunoblotting with the indicated antibodies. (B–D) HEK293T cells stably expressing FLAG-HA-Hook3 were generated, and extracts were examined by immunoblotting using antibodies against Hook3 (B). Anti-HA or control immune complexes from HEK293T/FLAG-HA-Hook3 cells were immunoblotted with the indicated antibodies (C) or eluted with HA peptide before trypsinization and LC/MS/MS analysis (D). The number of independent peptides as well as the total number of scans for these peptides is indicated, as well as the coverage of each protein.

FTS, Hook proteins, and p107^FHIP form a single major complex as assessed by gel filtration. (A) Effect of Hook1 depletion on the abundance of Hook2 and Hook3 proteins in complexes with FTS. Extracts were made from HEK293T/FLAG-HA-FTS cells transfected with the indicated siRNAs and subjected to immunoprecipitation with anti-HA antibodies. Blots were probed with the indicated antibodies. (B) Gel filtration analysis of FTS/Hook complexes in wild-type cells and in cells depleted of either FTS or Hook proteins. HeLa cells were transfected with the indicated siRNAs for 48 h before lysis (see Supplemental Figure S2 for depletion). Extracts were subjected to gel filtration by using a Superdex 200 column as described in Materials and Methods (B). Aliquots of fractions were analyzed by immunoblotting using the indicated antibodies. Anti-FTS antibodies were affinity purified. (C) Gel filtration analysis of FLAG–HA–FTS complexes purified from HEK293T cells. Complexes purified as described in were subjected to gel filtration using a Superdex 200 column, and fractions were analyzed by immunoblotting. (D) LC/MS/MS analysis of purified FLAG–HA–FTS complexes after gel filtration. FLAG–HA–FTS complexes purified as described in were subjected to gel filtration as described in B. Fractions corresponding to the peak of Hook/FTS proteins by immunoblotting (C) were precipitated with trichloroacetic acid, trypsinized, and subjected to LC/MS/MS analysis to identify p107^FHIP. The number of unique peptides and total scans for the relevant proteins are indicated.

Anatomy of the FHF complex. (A) A two-hybrid screen for proteins that interact with FTS by using a HeLa cells activation domain library was performed in PJ69-4A cells. Two Hook1 clones and one Hook3 clone were recovered. The location of the FTS-interacting clones on the Hook domain structure is shown. (B) Schematic of Hook1 fragments generated along with the sequence of the C-terminal FTS interacting region of Hook proteins and the results of binding studies. The positions of 2 helices in the C terminus of Hook1 (as determined using the JPRED secondary structure prediction tool; http://www.compbio.dundee.ac.uk/∼www-jpred/) are shown. The positions of mutations in helix 1 within the Hook1^4A mutant are indicated by “A” for alanine. (C) A C-terminal fragment of Hook1 is necessary and sufficient for interaction with FTS. HEK293T cells were transfected with vectors expressing the indicated proteins and after 48 h, cell extracts were generated and incubated with GSH-Sepharose. Bound proteins and control lysates were subjected to immunoblotting with the indicated antibodies. (D) The Hook1^4A mutant cannot bind FTS. Experiments were performed as described in C. (E) The Hook1^4A mutant maintains interaction with Hook2 and Hook3. HEK293T cells were transfected with vectors expressing the indicated proteins and after 48 h, cell extracts were generated and incubated with anti-FLAG (M2) resin. Bound proteins and control lysates were subjected to immunoblotting with the indicated antibodies. (F) A structure of UbcH5 bound to ubiquitin (PDB code: 2fuh), showing the residues in UbcH5 that correspond structurally to the W106/F107 and V121/F122 residues mutated in FTS, based on sequence alignments using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html). (G) FTS^W106AF107A fails to interact with Hook proteins. Plasmids expressing the indicated FLAG-tagged wild-type or mutant FTS proteins were transfected into HEK293T cells, and after 48 h, cell extracts were generated and incubated with anti-FLAG (M2) resin. Bound proteins and control lysates were subjected to immunoblotting with the indicated antibodies.

Depletion of FHF complex components reduces the ability of Vps18 to promote late endosome/lysosome clustering. (A) HeLa cells were transfected with control siRNA or siRNA targeting FTS. After 48 h, cells were transfected with a plasmid expressing GFP-Vps18, and 60 h later, late endosomal/lysosomal clusters were examined by immunofluorescence using anti-LAMP1 antibodies in conjunction with detection with Alexa598-conjugated secondary antibodies (red). GFP-Vps18 was identified by GFP fluorescence (green). To determine the integrated intensity for LAMP1 within clusters, a threshold (+ Threshold) was applied such that the maximal pixel signal was in the linear range. In the absence of threshold (− Threshold), individual vesicles not present within clusters can be seen in cells wherein FTS was depleted. Integrated intensities of Vps18 before thresholding (including both clustered Vps18 and dispersed Vps18), as well as LAMP1 aggregates after thresholding, are presented. The integrated intensities for LAMP1 and GFP-Vps18 are shown. The integrated intensities for GFP-Vps18 ranged from ∼20,000 to ∼40,000 over all the cells analyzed. (B) Quantification of the effects of FTS depletion on Vps18-mediated endosomal clustering. The integrated intensity of LAMP1 within GFP–Vps18-positive clusters was determined using MetaMorph software as described in Materials and Methods for 10–15 cells in each of three independent experiments (30–45 cells total). The mean ± SEM is indicated. Depletion of the indicated proteins displayed statistical significance using the F-test (***p < 0.001). (C–D) Immunoblotting of extracts from cells transfected with the indicated siRNAs as described in A. Extracts were separated by SDS-PAGE and blots probed with the indicated antibodies. The anti-FTS antibodies used here were affinity purified. To demonstrate depletion of p107^FHIP, cells were cotransfected with a vector expressing GST-p107^FHIP and extracts blotted with anti-GST antibodies.

Proteomic analysis of FTS complexes identifies a new complex containing Hook proteins and C11ORF56/p107^FHIP. (A) Phylogenetic tree of the human E2 conjugating family, generated using ClustalW. The variant E2 protein FTS lacking the active site cysteine residue found in catalytically active E2 enzymes is shown in bold. (B) Immunoblot analysis of extracts from HEK293T cells or HEK293T cells stably expressing FLAG-HA-FTS from an MSCV-based retrovirus by using anti-FTS antibodies. To demonstrate the specificity of the antibodies, cells were previously transfected with control siRNA or an siRNA targeting FTS. Blots were stripped and reprobed with actin as a loading control. (C and D) SDS-PAGE and mass spectral analysis of FLAG-HA-FTS complexes. Tandem anti-FLAG/anti-HA immune complexes from HEK293T cells or HEK293T/FLAG-HA-FTS cells separated on a 4–20% gradient SDS-PAGE gel and stained with silver (C). The indicated gel slices were analyzed by mass spectrometry (D). The number of independent peptides identified for each protein is shown. These proteins were not found in the negative control (lane 1). (E) LC/MS/MS analysis of tandem and single HA purified FLAG–HA–FTS complexes. Tandem (FLAG-HA) or single anti-HA purified complexes derived from four 15-cm dishes of cells were eluted with HA peptide, precipitated with trichloroacetic acid to remove the eluting peptide, trypsinized, and subjected to LC/MS/MS. Cells expressing FLAG-HA-GFP were used in parallel as a control. The number of independent peptides as well as the total number of scans for these peptides is indicated, as well as the coverage of each protein. (F) Extracts from HEK293T cells were subjected to immunoprecipitation using anti-Hook1, anti-Hook2, or anti-Hook3 antibodies (or anti-His tag antibodies as a negative control), and complexes were subjected to immunoblotting by using the indicated antibodies. Lysates (5% of input) were included in the blot. Note that under these conditions, the crude FTS antisera used in this experiment does not readily detect FTS in crude cell extracts, and FTS is highly enriched in the anti-Hook1 and Hook3 immune complexes. (G) HEK293T cells stably expressing FLAG-HA-Hook1 were generated and extracts examined by immunoblotting using antibodies against Hook1. (H) Anti-HA or control immune complexes from HEK293T/FLAG-HA-Hook1 cells were immunoblotted with the indicated antibodies, with lysate (5%) as a positive control. Note that under these conditions, the crude FTS antisera used in this experiment does not readily detect FTS in crude cell extracts, and FTS is highly enriched in the anti-Hook1 and Hook3 immune complexes. (I) HEK293T cells stably expressing FLAG-HA-Hook2 were generated and extracts examined by immunoblotting using antibodies against Hook2. (J) Anti-HA or control immune complexes from HEK293T/FLAG-HA-Hook2 cells were immunoblotted with the indicated antibodies, with lysate (5%) as a positive control. In this experiment, affinity-purified anti-FTS was used, which was capable of detecting FTS present in cell extracts (lane 1). (K) Two hybrid analysis of FTS and Hook proteins. The indicated open reading frames were cloned into either pDB or pAD vectors by using empty vectors as negative controls (see Materials and Methods) and transformed into PJ69-4A cells. Cells were plated on either Trp⁻, Leu⁻ media to select for plasmids or on Trp⁻, Leu⁻, His⁻, Ade⁻ to demonstrate the two hybrid interaction.