Abstract
Several protein families control intracellular transport processes in eukaryotic cells. Here, we show that the Rab11 GTPase effector protein Rab11-FIP3 (henceforth, FIP3) directly interacts with the dynein light intermediate chain 1 (DLIC-1, gene symbol DYNC1LI1) subunit of the cytoplasmic dynein 1 motor protein complex. We show that Rab11a, FIP3 and DLIC-1 form a ternary complex and that DLIC-1 colocalises with endogenous FIP3 and Rab11a in A431 cells. We demonstrate that association between FIP3 and DLIC-1 at the cell periphery precedes minus-end-directed microtubule-based transport, that FIP3 recruits DLIC-1 onto membranes, and that knockdown of DLIC-1 inhibits pericentrosomal accumulation of key endosomal-recycling compartment (ERC) proteins. In addition, we demonstrate that expression of a DLIC-1-binding truncation mutant of FIP3 disrupts the ability of ERC proteins to accumulate pericentrosomally. On the basis of these and other data, we propose that FIP3 links the Rab11 GTPase and cytoplasmic dynein to mediate transport of material from peripheral sorting endosomes to the centrally located ERC.
Highlights
Receptor-mediated endocytosis involves the internalisation of specific extracellular molecules following their binding to specific receptors located on the cell surface (Maxfield and McGraw, 2004)
To determine whether FIP3 can directly interact with the dynein motor complex, we used far-western blotting to investigate the ability of FIP3 to bind dynein light intermediate chain 1 (DLIC-1), a known cargo-binding subunit of the dynein motor complex (Bielli et al, 2001; Malone et al, 2003; Purohit et al, 1999; Sivaram et al, 2009)
Because we had identified a direct interaction between FIP3 and DLIC-1, and given that FIP3 is required for pericentrosomal positioning of the endosomal-recycling compartment (ERC) (Horgan et al, 2007), we considered it plausible that FIP3-dynein might mediate the trafficking of material from a peripheral location in the cell towards the pericentrosomal ERC
Summary
Receptor-mediated endocytosis involves the internalisation of specific extracellular molecules (ligands) following their binding to specific receptors located on the cell surface (Maxfield and McGraw, 2004). Material that enters cells by this means is typically delivered to a peripheral organelle with tubulovesicular morphology known as the sorting endosome (Maxfield and McGraw, 2004). Material can be returned to the cell surface either directly from peripheral sorting endosomes (the fast recycling pathway) or indirectly by accessing the endosomal-recycling compartment (ERC) (the slow recycling pathway). Members of the Rab GTPase subfamily [Rab11a, Rab11b and Rab11c ( known as Rab25)] are enriched on the cytosolic face of the ERC and have emerged as key regulators of membrane trafficking through this organelle (Schlierf et al, 2000; Ullrich et al, 1996). FIP3 appears to function during cell motility (Jing et al, 2009) and, as cells divide, FIP3 is involved in membrane delivery from the ERC to sites of membrane insertion on the cleavage furrow and/or midbody (Horgan et al, 2004; Wilson et al, 2005)
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