Rab11-FIP3 localises to a Rab11-positive pericentrosomal compartment during interphase and to the cleavage furrow during cytokinesis

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Abstract

The Rab11-family interacting protein 3 (Rab11-FIP3), also known as Arfophilin and Eferin, is a Rab11 and ADP-ribosylation factor (ARF) binding protein of unknown function. Here, we sought to investigate the subcellular localisation and elucidate the function of Rab11-FIP3 in eukaryotic membrane trafficking. Utilising a polyclonal antibody specific for Rab11-FIP3, we have demonstrated by immunofluorescence microscopy that Rab11-FIP3 colocalises with Rab11 in a distinctive pericentrosomal location in A431 cells. Additionally, we found that Rab11-FIP3 localises to punctate vesicular structures dispersed throughout A431 cells. We have demonstrated that both Rab11 and Rab11-FIP3 localise to the cleavage furrow during cytokinesis, and that Rab11-FIP3 localisation is dependent on both microtubule and actin filament integrity. We show that Rab11-FIP3 does not enter brefeldin A (BFA) induced membrane tubules that are positive for the transferrin receptor (TfnR). Furthermore, we show that expression of an amino-terminally truncated mutant of Rab11-FIP3 (Rab11-FIP3(244–756)) does not inhibit transferrin (Tfn) recycling in HeLa cells. It is likely that Rab11-FIP3 is involved in trafficking events other than Tfn trafficking; these may include the transport of endosomally derived membrane to the cleavage furrow during cytokinesis.

Section snippets

Materials and methods

cDNA cloning and plasmid construction. pGEX-3X/Rab11-FIP3(2–246) was constructed by subcloning a 0.8-kb BamHI fragment, corresponding to Rab11-FIP3(2–246), into pGEX-3X (Amersham Biosciences). pGFP2-C2/Rab11 was constructed by subcloning canine Rab11 from the previously described pLex-Rab11 construct [19], [20], as a 0.85-kb EcoRI–PstI fragment into pGFP2-C2 (kind gift from R. Pepperkok). pEGFP-C1/Rab11-FIP3(2–756) was constructed by subcloning the 2.4-kb EcoRI fragment from the previously

Polyclonal anti-Rab11-FIP3 antibody recognises endogenous Rab11-FIP3 in A431 and HeLa cells

Previous studies on Rab11-FIP3 and its biological significance have relied on the overexpression of protein. However, it is becoming increasingly apparent that the overexpression of the Rab11-FIPs causes a perturbation of the ERC morphology [11], [39]. Consequently, we sought to study endogenous Rab11-FIP3 through the generation of an antibody that specifically recognises this Rab11-FIP family member. To obtain a Rab11-FIP3 specific antibody, we chose an immunising peptide that represented

Discussion

The realisation that Rab11-FIP3 is identical to the previously described ARF interacting protein (Arfophilin) provides tantalising clues that this dual effector protein is likely to play a role in coordinating membrane trafficking and cytokinetic events. However, despite substantial two-hybrid and biochemical evidence supporting a role for Rab11-FIP3 as a Rab11 and ARF effector, little has been documented with respect to either the intracellular distribution of the endogenous protein or its

Acknowledgements

The authors are grateful to T. Nagase (Kazusa DNA Research Institute, Japan) for the cDNA encoding Rab11-FIP3 (KIAA0665); C. D’Souza-Schorey (University of Notre Dame, Indiana, USA) for the ARF6 cDNA; A. Hendrick (University of Cambridge, UK) for the ARF4 and -5 constructs; R. Pepperkok (EMBL Heidelberg, Germany) for the GFP2 plasmids; J. Gruenberg (University of Geneva, Switzerland) for the anti-LBPA antibody; and A. Lindsay for the anti-Rab11 and anti-RCP antibodies. The authors also wish to

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    These authors contributed equally to this work.

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