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First published online 10 August 2004
doi: 10.1242/jcs.01280

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The C2 domains of the class I Rab11 family of interacting proteins target recycling vesicles to the plasma membrane

Molecular Cell Biology Laboratory, Department of Biochemistry, Biosciences Institute, University College Cork, Cork, Ireland

View larger version (72K): [in a new window]   Fig. 1. (A) Schematic representation of the class I Rab11-FIPs, indicating the location of the C2 domain (C2), the -helical coil (C-C) and the Rab-binding domain (RBD). (B) HeLa cells expressing the indicated GFP fusion protein. Truncation mutants lacking the N-terminal homology domain (NHD) form large vesicular structures. Scale bar, 10 µm.

View larger version (53K): [in a new window]   Fig. 2. The C2 domains of the class I Rab11-FIPs bind PtdIns(3,4,5)P3 and PA. (A) PipArraysTM were overlayed with the indicated GST fusion proteins and immunoblotted with anti-GST antibody. (B) Densitometry of the PipArrays indicating that the class I Rab11-FIPs bind preferentially to PtdIns(3,4,5)P3. The results are the mean of three independent experiments. (C) Nitrocellulose was spotted with serial dilutions of PS, PA, PC and PE. They were subsequently overlayed with the indicated GST fusion proteins and immunoblotted with anti-GST antibody. (D) Densitometry of the protein-lipid assay, indicating a preference for PA. The results are representative of three independent experiments.

View larger version (30K): [in a new window]   Fig. 3. ClustalW alignment of the NHD of the class I Rab11-FIPs. The line indicates the conserved C2 domain. The red boxes mark the conserved lysines and arginines in the region downstream of the C2 domain.

View larger version (79K): [in a new window]   Fig. 4. Endogenous RCP and Rab11-FIP2 translocate to the plasma membrane upon PtdIns(3,4,5)P3 and PA synthesis in A431 cells. (A) Cells were unstimulated, treated with 100 ng ml–1 EGF, for 20 minutes at 37°C, or pretreated with 100 nM wortmannin before incubation with EGF. The cells were then fixed and processed for immunofluorescence with antibodies to RCP or Rab11-FIP2. (B) Cells were unstimulated, treated with 1 µM PMA for 30 minutes at 37°C or pretreated with 100 nM wortmannin followed by processing for immunofluorescence as above. (C) Cells were either treated with DMSO or 1 µM PMA for 30 minutes at 37°C before fixation. The cells were then stained for RCP (green) and ZO-1 (red). Scale bar, 10 µm.

View larger version (51K): [in a new window]   Fig. 5. The NHD is essential for plasma membrane translocation. A431 cells expressing the indicated GFP-fusion protein were treated with either 1 µM PMA or with DMSO alone for 30 minutes at 37°C. Cells were fixed and analysed by confocal microscopy. The fusion proteins lacking the NHD failed to translocate to the plasma membrane in cells stimulated with PMA. Scale bar, 10 µm.

View larger version (116K): [in a new window]   Fig. 6. The NHD alone of the class I Rab11-FIPs can localize to membranes in stimulated cells. A431 cells transiently transfected with the indicated constructs were untreated, stimulated with 100 ng ml–1 EGF for 20 minutes at 37°C or stimulated with 1 µM PMA for 30 minutes at 37°C before fixation. The fusion proteins localized to the plasma membrane in stimulated cells (white arrows). In PMA-treated cells, the fusion proteins also displayed extensive labelling of intracellular membranes (red arrows). Scale bar, 10 µm.

View larger version (104K): [in a new window]   Fig. 7. (A) Rab11 translocates to the plasma membrane with class-I Rab11-FIPs. A431 cells expressing GFP-Rip11 were treated with either solvent or 1 µM PMA for 30 minutes at 37°C. The cells were then fixed and labelled with an antibody to Rab11. In unstimulated cells, GFP-Rip11 and Rab11 colocalize on intracellular vesicles and to the ERC. In PMA-treated cells, they display extensive colocalization at the plasma membrane. (B) A431 cells were treated with DMSO solvent alone or with 1 µM PMA for 30 minutes at 37°C. The cells were then fixed and labelled for RCP and the TfnR. In contrast to RCP-positive vesicles, TfnR-positive vesicles do not translocate to the plasma membrane. Scale bar, 10 µm.

View larger version (120K): [in a new window]   Fig. 8. The class I Rab11-FIPs localize with Akt to the plasma membrane in EGF-treated cells. A431 cells co-expressing HA-Akt and the indicated GFP-fused class I Rab11-FIP were treated with 100 ng ml–1 EGF for 20 minutes at 37°C. The cells were fixed and labelled with an anti-HA antibody. The insets display regions of colocalization at the plasma membrane. Scale bar, 10 µm.