Volume 8 issue 10 October 2007
Rab6-interacting protein1 (Rab6IP1) links Rab6 and Rab11 function
S Miserey-Lenkei, F Waharte, A Boulet, M-H Cuif, D Tenza, A El Marjou, G Raposo, J Salamero, L Héliot, B Goud and S Monier

Supplemental Figure 1: R6IP1 is a cytosolic protein. Post-nuclear supernatants (PNS) were prepared from mouse brain (lane a) and from HeLa cells (lane b). HeLa cell PNS (lane c) was then centrifuged at 100,000 x g in order to sort membranes (mb, lane e) from the cytosol (lane d). Western blotting was performed using a polyclonal rabbit antiserum generated against recombinant R6IP1A purified from baculovirus-infected cells. The antibody recognized in mouse PNS (lane a), a major band migrating at an apparent molecular mass of 130 kDa, corresponding to the predicted size of the protein. In HeLa cell PNS (lane b), the antibody stained additional bands. They likely correspond to unrelated proteins since they were still present after silencing of R6IP1 by siRNAs (data not shown). Molecular weight markers are shown on the right.

Figure 1 (.jpg)

Supplemental Figure 2: Localization of the Golgi apparatus in CFP-Rab11A and YFP-R6IP1A co-expressing cells. Cells transfected with CFP-Rab11A (green) and YFP-R6IP1A (red) (corresponding to Fig. 4C) were stained for endogenous Rab6 (blue). Overlay is shown on the right. Scale bar, 10 µm.

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Supplemental Figure 3: Quantification of subcellular localization of GFP-R6IP1A/B. The relative distribution of GFP-R6IP1A and GFP-R6IP1B in transfected HeLa cells was evaluated by analyzing directly under the electron microscope selected cell profiles from two distinct grids. A total of 764 and 695 gold particles were counted for GFP-R6IP1A and GFP-R6IP1B, respectively and assigned to the compartment over which they were located. The definition of the distinct compartments was based on their morphology and their previous characterization by immunogold labeling with organelle markers (TGN46 for the TGN, LAMP-1 for late endosomes/ lysosomes) and accessibility to internalized Transferrin-HRP (endosomes). Tubulovesicular membranes that were located at the trans side of the Golgi were considered as TGN. Endosomes were defined as electron-lucent vacuoles with no internal membranes or few internal vesicles and tubulovesicular elements closely apposed to the vaccuoles. MVBs were compartments delimited by a membrane with numerous internal vesicles. Electron-dense compartments with no or few internal membranes were classified as lysosomes. Other vesicular structures dispersed in the cytoplasm with no assigned origin were considered as « cytoplasmic vesicles ». Labeling of mitochondria with anti-GFP antibodies was less than 0.1%.

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Supplemental Figure 4: Localization of GFP-R6IP1A and endogenous Rab6 (a, c) and Rab11 (b, d) in metaphase (a, b) and cytokinesis (c, d). Cells were transfected for 24 h with a plasmid encoding GFP-R6IP1A and then processed for immunofluorescence. Cells were labeled with antibodies against Rab6 (red), Rab11 (red) and β-tubulin (blue). In C, images were deconvolved for better visualization of Rab6 positive structures in the intracellular bridge (arrowheads). Overlays are shown on the right. Scale bars, 10 µM. Similar localization was found for GFP-R6IP1B (data not shown).

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Supplemental Table 1: Yeast two-hybrid assays.

Supplemental Table 2: Statistics of transferrin recycling experiments (Figure 9; p of Student's t tests).

Supplemental Movies: HeLa cells treated with control (movie 1) or R6IP1 siRNA (movie 2) were imaged for 72h by time-lapse phase contrast videomicroscopy. Some R6IP1 siRNA treated cells (movie 2, arrow) entered mitosis normally, rounded up, but were subsequently blocked in mitosis and died after several hours. Details of a R6IP1 siRNA treated cell undergoing a metaphase arrest (movie 3, arrow); note the alignment of chromosomes at the metaphase plate during the metaphase block (arrowhead). Details of a cell (arrow) undergoing cytokinesis defect after R6IP1 siRNA (movie 4). The numbers correspond to the time in minutes after the beginning of the recording.

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