Characterization of RIN3 as a guanine nucleotide exchange factor for the Rab5 subfamily GTPase Rab31

Abstract

The small GTPase Rab5, which cycles between GDP-bound inactive and GTP-bound active forms, plays essential roles in membrane budding and trafficking in the early endocytic pathway. Rab5 is activated by various vacuolar protein sorting 9 (VPS9) domain-containing guanine nucleotide exchange factors. Rab21, Rab22, and Rab31 (members of the Rab5 subfamily) are also involved in the trafficking of early endosomes. Mechanisms controlling the activation Rab5 subfamily members remain unclear. RIN (Ras and Rab interactor) represents a family of multifunctional proteins that have a VPS9 domain in addition to Src homology 2 (SH2) and Ras association domains. We investigated whether RIN family members act as guanine nucleotide exchange factors (GEFs) for the Rab5 subfamily on biochemical and cell morphological levels. RIN3 stimulated the formation of GTP-bound Rab31 in cell-free and in cell GEF activity assays. RIN3 also formed enlarged vesicles and tubular structures, where it colocalized with Rab31 in HeLa cells. In contrast, RIN3 did not exhibit any apparent effects on Rab21. We also found that serine to alanine substitutions in the sequences between SH2 and RIN family homology domain of RIN3 specifically abolished its GEF action on Rab31 but not Rab5. We examined whether RIN3 affects localization of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is transported between trans-Golgi network and endocytic compartments. We found that RIN3 partially translocates CD-MPR from the trans-Golgi network to peripheral vesicles and that this is dependent on its Rab31-GEF activity. These results indicate that RIN3 specifically acts as a GEF for Rab31.

FIGURE 1.

Cell-free GEF activity of the RIN proteins and Rabex-5 for Rab5 proteins. The Rab5 proteins (A, GST-Rab5, Rab21, and Rab31) and GEF proteins (B, FLAG-tagged RIN1–3 and Rabex-5) purified from E. coli and baculovirus-infected Sf9 cells, respectively, were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. GST-Rab5 (C, 6 pmol of alive GTPγS binding activity), Rab21 (D, 4 pmol), or Rab31 (E, 6 pmol) were incubated at 30 °C with 1 μm [³⁵S]GTPγS for the indicated times in the absence (FLAG peptide alone) and presence of 8 pmol of FLAG-Rabex-5 (filled circles), RIN1 (filled squares), RIN2 (filled diamonds), or RIN3 (filled triangles). The amounts of [³⁵S]GTPγS bound to Rab5 proteins at each time point are presented. No [³⁵S]GTPγS binding activity was detected in the fractions containing RIN proteins or Rabex-5 (data not shown). The data obtained from three independent experiments are shown with mean ± S.E. ***, p < 0.001; **, p < 0.01; *, p < 0.05 versus Rab alone samples.

FIGURE 2.

In cell GEF activity of the VPS9 domain proteins for the Rab5 proteins. A, HEK293T cells expressing myc-Rab31 and FLAG-mock, RIN1–3, or Rabex-5 were metabolically radiolabeled with ³²P_i for 4 h. Myc-tagged Rab31 was immunoprecipitated with an anti-myc monoclonal antibody, and nucleotides associating with Rab31 were separated by thin layer chromatography (top). The radioactivity of GTP and GDP was quantified, and the percentages (%) of GTP-bound Rab31 are shown in the bottom lanes. Immunoprecipitated samples (middle) and total lysates (bottom) from the radiolabeled cells were separated by SDS-PAGE and immunoblotted with anti-myc and anti-FLAG antibodies, respectively. Asterisk (*) shows a nonspecific band. B, HEK293T cells expressing myc-Rab5 subfamily (Rab5, 21, and 31) and the FLAG-VPS9 domain proteins (mock, RIN1, RIN2, RIN3, Rabex-5, Gapex-5, ALS2, ALS2CL, and Varp) were metabolically radiolabeled with ³²P_i, and the percentages (%) of GTP-bound Rab GTPases were determined as described in A. The data obtained from more than three independent experiments are shown with mean ± S.E. (error bars).

FIGURE 3.

Mutations in the VPS9 domains of RIN proteins impair their GEF activities for Rab31. A and B, myc-Rab31 was cotransfected with wild-type (WT), DP_AA, or YT_AA mutant of FLAG-RIN1–3 into HEK293T cells. Cells were metabolically radiolabeled with ³²P_i, and the percentages (%) of GTP-bound Rab31 were determined (top) as shown in Fig. 2. Immunoprecipitated samples (middle) and total lysates (bottom) from the radiolabeled cells were also separated by SDS-PAGE and immunoblotted with anti-myc and anti-FLAG antibodies, respectively. The data obtained from three independent experiments are shown (B) with mean ± S.E. (error bars). ***, p < 0.001; **, p < 0.01 versus mock-transfected cells. C, wild-type and YT_AA mutant versions of FLAG-RIN3 were purified from baculovirus-infected Sf9 cells. Recombinant proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. D, GST-Rab31 (4 pmol of alive GTPγS binding activity) was incubated at 30 °C with 1 μm [³⁵S]GTPγS for the indicated times in the presence of wild-type (filled squares) and YT_AA mutant (filled triangles) of FLAG-RIN3 (8 pmol) for 90 min. The data obtained from three independent experiments are shown with mean ± S.E.

FIGURE 4.

Expression of RIN3 induces the enlargement of Rab31-positive endosomes and tubulovesicular structures. A, EGFP-Rab31 (right), wild-type (center), and YT_AA mutant (right) versions of DsRedm-RIN3 were transiently transfected into HeLa cells. B–D, EGFP-Rab31 was cotransfected with DsRedm-mock (B), wild-type (C), and YT_AA mutant (D) versions of RIN3 in HeLa cells. Merged images of the two signals are displayed in the right panels, and insets in C and D are magnifications of the areas highlighted by white squares. Scale bars: 10 μm.

FIGURE 5.

Characterization of Rab31-positive enlarged endosomes and tubulovesicular structures induced by RIN3. EYFP-RIN3 was transiently transfected with ECFP-Rab31 into HeLa cells. After incubation for 48 h, transfected cells were immunostained with anti-EEA1 (A), transferrin receptor (Tfn R, B), Lamp1 (C), and TGN46 (D) antibodies, and merged images of the three signals are displayed in the right panels. Scale bars: 10 μm.

FIGURE 6.

RIN3/S_A mutant specifically attenuates interaction with, and GEF activity for, Rab31. A, analysis of RIN3-Rab31 interactions using the yeast two-hybrid system. Two-hybrid bait plasmids encoding mock (white), RIN3/WT (black), and RIN3/S_A (gray) were cotransformed into the Y190 yeast strain with the Rab5 or Rab31 prey plasmid. Cotransformants were subjected to the β-galactosidase assay. The data obtained from three independent experiments are shown with the mean ± S.E. (error bars). B and C, The purified GST-Rab5 (B) or Rab31 (C) (4 or 5 pmol of alive GTPγS binding activity, respectively) were incubated at 30 °C with 1 μm [³⁵S]GTPγS for the indicated times in the presence of FLAG-RIN3/WT (filled squares), RIN3/S_A (filled triangles), or FLAG peptide alone (open circles) (each 8 pmol). D, in cell GEF activities of FLAG-mock (white), RIN3/WT (black), and RIN3/S_A (gray) for Rab5 or Rab31 in HeLa cells were determined as shown in Fig. 2. E–I, GFP-Rab5 (E and F) or Rab31 (G and H) were cotransfected with FLAG-RIN3/WT (E and G) or RIN3/S_A (F and H) in HeLa cells and immunostained with anti-FLAG antibody. The degrees of colocalization of 25 cells were evaluated by cross-correlation analyses and are shown as Pearson's coefficient (I). ***, p < 0.001. Error bars, S.E.

FIGURE 7.

Intracellular transport of the mannose 6-phosphate receptor from TGN to early endosome is enhanced by RIN3. A, HeLa cells expressing CD-MPR-EGFP were immunostained with anti-TGN46 antibody. B–E, CD-MPR-EGFP was cotransfected with DsRedm-mock (B), RIN3/WT (C), RIN3/YT_AA (D), and RIN3/S_A (E) in HeLa cells. Merged images of the two signals are displayed in the right.