Fig. 2. Interaction of GGA-GAE with the Rabaptin-5–Rabex-5 complex. (A) Bovine brain cytosol was subjected to four consecutive incubations with anti-Rabaptin-5 immobilized on protein G–Sepharose. Samples of the untreated cytosol (round 0) and of the materials collected after each round of consecutive immunodepletions (rounds 1–4) were analyzed by SDS–PAGE and immunoblotting (IB) with anti-Rabaptin-5 or anti-Rabex-5 antibodies (blots on the left). The cytosol fractions corresponding to rounds 0–4 were also used in pull-down experiments with GST–GGA1-GAE immobilized to glutathione– Sepharose. The material pulled-down by the GST–GGA1-GAE was analyzed by SDS–PAGE and immunoblotting (blots on the right). (B) Bovine brain cytosol was subjected to four consecutive rounds of treatment with either GST–GGA1-GAE or GST immobilized to glutathione–Sepharose. The control cytosol (round 0) and the fractions corresponding to rounds 1–4 were analyzed by SDS–PAGE and immunoblotting.

Fig. 4. Different regions of Rabaptin-5 are responsible for interaction with GAT and GAE domains of GGAs. The AH109 yeast strain was co-transformed with the indicated constructs (GGA domains fused to the GAL4 binding domain and Rabaptin-5 fragments fused to the GAL4 activation domain). (A) The various Rabaptin-5 constructs that were assayed. After selection, co-transformants were plated on medium without histidine (–His), to monitor HIS3 reporter gene activation due to interaction of fusion proteins, and on medium containing histidine (+His) to control growth/loading of the co-transformants (B). The –His plates were supplemented with 30 mM 3-AT (a competitive inhibitor of the His3 protein) to suppress background growth and minimize non-specific interactions between constructs.

Fig. 6. Alanine scan mutagenesis in Rabaptin-5 (5–476) defines a GGA-GAE-binding sequence: FGXLV. (A) Sequence of the Rabaptin-5 (435–447) segment. (B and C) Interaction of GGA-GAE domains with Rabaptin-5 (5–476) mutants: either blocks of 3–4 contiguous amino acids corresponding to codons 435–447 (B) or individual residues in the 438–443 region (C) were introduced into Rabaptin-5 (5–476). Experiments were performed as indicated in the legend to Figure 5, with the exception that the co-transformants were plated on –His plus 5 mM 3-AT, –His medium, and –His medium plus 15 mM 3-AT for evaluation of interactions with GAE domains of GGA1, GGA2 and GGA3, respectively. (D) The consensus secondary structure prediction for Rabaptin-5 (Network Protein Sequence Analysis of the Pole Bio-informatique Lyonnais), as well as the position of the GGA-GAE-binding sequence.

Fig. 8. Binding of biotinylated Rabaptin-5 peptides to γ-adaptin ear and GGA-GAE domains. Both a biotinylated peptide corresponding to the Rabaptin-5 (435–447) sequence (A) and a variant with F439A and L442A substitutions (B) were immobilized on streptavidin–agarose and tested for their ability to bind GST fusions to the γ1- and γ2-adaptin ear and GGA1-GAE domains (GST and GST–β3A were used as negative controls). The material bound to the immobilized peptides was analyzed by SDS–PAGE and immunoblotting (IB) using anti-GST antiserum (A and B). The Coomassie Blue staining of a gel loaded with the various GST fusions is depicted in (C).

Fig. 10. Changes in the intracellular distribution of endogenous GGA1, CI-MPR and TfR upon expression of GFP–Rabaptin-5 in HeLa cells. (A–C) Confocal immunofluorescence microscopy showing discrete areas of co-localization of endogenous Rabaptin-5 and GGA-1 in untransfected HeLa cells. HeLa cells were subjected to a brief permeabilization pulse immediately before fixation with 4% formaldehyde. Fixed cells were subjected to double staining using mouse monoclonal anti-Rabaptin-5 and rabbit anti-GGA1 antibodies, followed by incubation with Cy3-conjugated anti-rabbit and Alexa 488-conjugated anti-mouse antisera. Scale bar = 10 µm. (D–L) Localization of endogenous GGA1 (D–F), CI-MPR (G–I) and TfR (J–L) to enlarged endosomes (∼2 µm) induced by transient overexpression of GFP–Rabaptin-5 in HeLa cells. Fixed cells were subjected to double immunostaining using: rabbit anti-GGA1 and mouse monoclonal anti-GFP, followed by Cy3-conjugated anti-rabbit and Alexa 488-conjugated anti-mouse secondary antisera (D–F); mouse monoclonal anti-CI-MPR and rabbit anti-GFP, followed by Alexa 568-conjugated anti-mouse and Alexa 488-conjugated anti-rabbit antisera (G–I); and mouse monoclonal anti-TfR and rabbit anti-GFP followed by Alexa 568-conjugated anti-mouse and Alexa 488-conjugated anti-rabbit antisera (J–L). The use of anti-GFP antisera allowed a more sensitive detection of cells expressing low levels of GFP–Rabaptin-5 than direct detection of GFP fluorescence. Scale bar = 10 µm.

Fig. 1. Binding of GST fusion proteins to Rabaptin-5 and Rabex-5 from bovine brain cytosol. (A and B) The domains and relevant motifs in human GGA1 and human Rabaptin-5 respectively. The Rabaptin-5 scheme shows the Rab4- and Rab5-binding domains along with a DPF motif (codons 388–390) and the four regions predicted to form coiled-coil structures (C1-1, C1-2, C2-1 and C2-2). Various GST fusion proteins were immobilized on glutathione–Sepharose and subsequently incubated with bovine brain cytosol. Bound proteins were subjected to SDS–PAGE and immunoblotting (IB) using a mouse monoclonal antibody to Rabaptin-5 (C and D) or a rabbit polyclonal antiserum to Rabex-5 (E). (D) Blots from a separate experiment where membranes were overexposed to allow comparison of signals corresponding to GAE, hinge and hinge + GAE constructs of GGA1 and GGA2. (F) The Coomassie Blue staining of a gel loaded with the various GST fusion proteins. The first lanes in (C) and (E) show the signals given by 2% of the cytosol used in the binding step. The positions of molecular mass markers (in kDa) are indicated on the left.

Fig. 3. Co-immunoprecipitation of GGAs and Rabaptin-5. (A) HeLa cell lysates were subjected to immunoprecipitation (IP) using either rabbit anti-GGA1, mouse monoclonal anti-Rabaptin-5, mouse monoclonal anti-GFP or a rabbit antiserum against the σ subunit of AP-3 (anti-σ3). The immunoprecipitated materials were analyzed subsequently by SDS–PAGE and immunobotting (IB) with mouse monoclonal anti-Rabaptin-5. (B and C) Lysates from HeLa cells transiently transfected with the indicated constructs were subjected to IP using mouse monoclonal anti-GFP followed by SDS–PAGE and IB with anti-Rabaptin-5 (B). Samples of the lysates were also analyzed by SDS–PAGE and immunoblotting with anti-GFP, in order to assess the levels of expression of the different constructs (C).

Fig. 5. C-terminal coiled-coils in Rabaptin-5 bind the GAT domains of GGA1 and GGA2, while residues in the Rabaptin-5 428–455 region are important for interaction with the GGA-GAE domain. (A) The interaction between deletion mutants of the Rabaptin-5 C-terminal region and GAT domains of GGA1 and GGA2. (B) The interaction of an additional set of deletion mutants in the N-terminal half of Rabaptin-5 with GGA-GAE domains. Also shown in (B) is the interaction with a Rabaptin-5 (5–546) mutant containing a DPF388–390AAA substitution. Experiments were performed as indicated in the legend to Figure 4, with the exception that co-transformants were plated on –His medium with or without 30 mM (A) or 15 mM 3-AT (B). The ++, + or – symbols indicate the relative strength of interactions between GGA domains and Rabaptin-5 mutants.

Fig. 7. Definition of domains important for the γ-adaptin–Rabaptin-5 interaction. (A) Different Rabaptin-5 fragments (schematized in Figures 4 and 5) were used to study the interactions with full-length γ1, γ1 and γ2 ears and GAE domains of GGA2 and GGA3 by yeast two-hybrid analysis. (B) Interaction between γ-adaptin ears and Rabaptin-5 (5–476) mutants. Co-transformants were plated on +His medium and on –His medium with the indicated concentrations of 3-AT.

Fig. 9. Rabaptin-5 interferes with GGA–clathrin interactions. (A) The effects of increasing concentrations of His₆-Rabaptin-5 (5–234) and His₆-Rabaptin-5 (5–546) on the binding of clathrin from bovine brain cytosol to GST–GGA2-hinge + GAE were assessed in a pull-down interference assay. Specificity controls include pull-down incubations with GST (left lane) as well as the effects of increasing concentrations of gelatin or BSA. (B) A mutant His₆-Rabaptin-5 (5–476) fragment containing three alanine substitutions in codons 438–440 (5–476*) does not affect the binding of clathrin to GST–GGA2-hinge + GAE. (C) Coomassie Blue staining of gels analyzing the preparations of His₆-tagged Rabaptin-5 fragments 5–234, 5–546 and 5–476*. The mobility of the full-length His₆-Rabaptin-5 (5–546) and 5–476* fragments is indicated by dotted lines on the left and right of the panel, respectively; preparations of these two fragments contained two proteolytic fragments recognized by anti-Rabaptin-5 in addition to the full-length protein.