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First published online September 12, 2003
doi: 10.1242/10.1242/jcs.00718

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RIN3: a novel Rab5 GEF interacting with amphiphysin II involved in the early endocytic pathway

Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan

View larger version (50K): [in a new window]   Fig. 1. The domain structure of the RIN members and northern blot analysis of RIN3 mRNA in human tissues. (A) Diagram of the structural features of the RIN-family members. The numbers at the bottom represent the amino acid residues. (B, top) The probe corresponding to the 729-base RIN3 cDNA was labelled by random priming and hybridized to a human multiple tissue RNA blot containing 2 µg lane-1 of poly(A) mRNA from various human tissues. (B, bottom) The northern blot was also performed with actin cDNA as a control.

View larger version (30K): [in a new window]   Fig. 2. RIN3 and RIN2 act as GEFs and stabilizers for Rab5b. (A) Flag-tagged RIN3, RIN2 (left) and prenylated Rab5b (right) purified from baculovirus-infected Sf9 cells were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. (B) The purified Rab5b (18 nM) that had been preloaded with 5 µM [3H]GDP was subjected to the nucleotide exchange assay in the presence of 300 nM RIN3 (triangles), RIN2 (squares) and Flag-peptide alone (circles). At the indicated times, an aliquot was removed from the reaction mixture, quenched and filtered through a nitrocellulose membrane. The membranes were subjected to liquid scintillation counting. The proportions of [3H]GDP retained in the membranes are presented as a function of the incubation times. (C) The purified Rab5b (18 nM) was incubated at 30°C with 1 µM [35S]GTPS in the presence of 300 nM RIN3 (triangles), RIN2 (squares) and Flag-peptide alone (circles). The amounts of [35S]GTPS bound to Rab5b are presented as a function of the incubation times. [35S]GTPS-binding activity was not detected in the fraction of RIN3 or RIN2 (data not shown). (D) Lysate was prepared from COS7 cells that had been transfected with Flag-RIN3 and immunoprecipitated with the anti-Flag antibody-conjugated resin. The resin was washed and incubated with or without GDP- or GTPS-bound Rab5b. Proteins bound to the resin were separated by SDS-PAGE and immunoblotted (IB) with the anti-Flag (top) and anti-Rab5 (bottom) antibodies.

View larger version (57K): [in a new window]   Fig. 3. RIN3 transfected into HeLa cells co-localizes with Rab5 but not EEA1. (A) HeLa cells were transiently transfected with RFP-RIN3 and cultured for 48 hours. The cells were fixed and labelled with antibodies to Rab5 (top) or EEA1 (bottom). The fluorescence of RFP-RIN3 (left) and Alexa-488 secondary antibody (middle) was visualized by confocal microscopy, and merged images of the two signals are displayed in yellow (right). Arrowheads indicate the co-localization of RIN3 and Rab5. (B) HeLa cells transiently expressing RFP-RIN3 and GFP-Rab5b were subjected to confocal microscopy, and the fluorescence of RFP-RIN3 (left) and GFP-Rab5b (middle) was visualized by confocal microscopy as described in (A). (C) HeLa cells transiently expressing the full-length (FL, left), N-terminal (N, middle) or C-terminal (C, right) form of RFP-RIN3 were subjected to confocal microscopy. (D) HeLa cells transiently expressing RFP-RIN1 (left) or RFP-RIN3 (right) were subjected to confocal microscopy. (E) Lysates from the transfected cells (RFP-RIN1, left; RFP-RIN3, right) were separated by SDS-PAGE and immunoblotted (IB) with an anti-RFP antibody. Scale bars, 10 µm.

View larger version (99K): [in a new window]   Fig. 4. Endocytic transferrin is transported through RIN3-positive vesicles to early endosomes in HeLa cells. HeLa cells expressing RFP-RIN3 were pulse-chased with Alexa-488/transferrin at 4°C, shifted to 25°C to allow its internalization and further incubated for the indicated times. The fluorescence of RFP-RIN3 (left) and transferrin (middle) was visualized by confocal microscopy, and merged images of the two signals are displayed in yellow (right). Arrowheads indicate the co-localization of RIN3 and transferrin. Scale bars, 10 µm.

View larger version (57K): [in a new window]   Fig. 5. The N-terminal region of RIN3 containing PRDs specifically interacts with the SH3 domain of amphiphysin II. (A) The full-length (FL) or SH3 domain (SH3) of GST-fused amphiphysin II (GST-amph II) or GST alone was incubated with Flag-RIN3 purified from baculovirus-infected Sf9 cells and glutathione resin. Proteins bound to the resin were separated by SDS-PAGE and immunoblotted (IB) with anti-Flag (top) and anti-GST (bottom) antibodies. (B) Lysates were prepared from HeLa cells expressing RIN3 and the full length (FL) or SH3-domain-deleted form (SH3) of Flag-tagged amphiphysin II (Flag-amph II) and incubated with glutathione resin. Proteins bound to the resin were separated by SDS-PAGE and immunoblotted with anti-RIN3 (top) and anti-Flag (bottom) antibodies. (C) Lysates were prepared from HeLa cells expressing the full length (FL), N-terminal (N) or C-terminal (C) form of Flag-tagged RIN3 (Flag-RIN3) and incubated with GST-fused amphiphysin II (GST-amph II) and glutathione resin. Proteins bound to the resin (GST-pull down) and the total lysate were separated by SDS-PAGE and immunoblotted with anti-Flag (top) and anti-GST (bottom) antibodies. (D) Flag-RIN1 and RIN3 were purified from baculovirus-infected Sf9 cells, and GST pull-down assay was performed as described in (A). Proteins bound to the resin (GST pull-down) and the total lysate were separated by SDS-PAGE and immunoblotted with anti-Flag (top) and anti-GST (bottom) antibodies.

View larger version (54K): [in a new window]   Fig. 6. Cytoplasmic amphiphysin II translocates into RIN3-positive vesicles in HeLa cells. (A,B) HeLa cells were transiently transfected with GFP-amphiphysin II (A) or GFP-amphiphysin II/SH3 (B) and RFP-mock (A,B, top) or RFP-RIN3 (A,B, bottom) and further incubated for 48 hours. The fluorescence of RFP (left) and GFP (centre) was visualized by confocal microscopy, and merged images of the two signals are displayed in yellow (right). (C) HeLa cells were transiently transfected with RFP-RIN3 (left), GFP-amphiphysin II (middle) and YFP-Rab5 (right), and further incubated for 48 hours. Scale bars, 10 µm.