The dual Rab11/Arf binding proteins, family of Rab11-interacting proteins FIP3 and FIP4 function in the delivery of recycling endosomes to the cleavage furrow and are, together with Rab11, essential for completion of abscission, the terminal step of cytokinesis. Here, we report that both FIP3 and FIP4 bind Arf6 in a nucleotide-dependent manner but exhibit differential affinities for Rab11 and Arf6. Both FIP3 and FIP4 can form ternary complexes with Rab11 and Arf6. Arf6 is localised to the furrow and midbody and we show that Arf6-GTP functions to localise FIP3 and FIP4 to midbodies during cytokinesis. Exo70p, a component of the Exocyst complex, also localises to the furrow of dividing cells and interacts with Arf6. We show that depletion of Exo70p leads to cytokinesis failure and an impairment of FIP3 and Rab11 localisation to the furrow and midbody. Moreover, Exo70p co-immunoprecipitates FIP3 and FIP4. Hence, we propose that FIP3 and FIP4 serve to couple Rab11-positive vesicle traffic from recycling endosomes to the cleavage furrow/midbody where they are tethered prior to fusion events via interactions with Arf6 and the Exocyst.

Dysfunctions of primary cilia and cilia-derived sensory organelles underlie a multitude of human disorders, including retinal degeneration, yet membrane targeting to the cilium remains poorly understood. Here, we show that the newly identified ciliary targeting VxPx motif present in rhodopsin binds the small GTPase Arf4 and regulates its association with the trans-Golgi network (TGN), which is the site of assembly and function of a ciliary targeting complex. This complex is comprised of two small GTPases, Arf4 and Rab11, the Rab11/Arf effector FIP3, and the Arf GTPase-activating protein ASAP1. ASAP1 mediates GTP hydrolysis on Arf4 and functions as an Arf4 effector that regulates budding of post-TGN carriers, along with FIP3 and Rab11. The Arf4 mutant I46D, impaired in ASAP1-mediated GTP hydrolysis, causes aberrant rhodopsin trafficking and cytoskeletal and morphological defects resulting in retinal degeneration in transgenic animals. As the VxPx motif is present in other ciliary membrane proteins, the Arf4-based targeting complex is most likely a part of conserved machinery involved in the selection and packaging of the cargo destined for delivery to the cilium.

Summary Sorting and recycling of endocytosed proteins are required for proper cellular function and growth. Internalized receptors either follow a fast constitutive recycling pathway, returning to the cell surface directly from the early endosomes, or a slow pathway that involves transport via perinuclear recycling endosomes. Slow recycling pathways are thought to play a key role in directing recycling proteins to specific locations on cell surfaces, such as the leading edges of motile cells. These pathways are regulated by various Rab GTPases, such as Rab4 and Rab11. Here we characterize the role of Rip11/FIP5, a known Rab11-binding protein, in regulating endocytic recycling. We use a combination of electron and fluorescent microscopy with siRNA-based protein knockdown to show that Rip11/FIP5 is present at the peripheral endosomes, where it regulates the sorting of internalized receptors to a slow recycling pathway. We also identify kinesin II as a Rip11/FIP5-binding protein and show that it is required for directing endocytosed proteins into the same recycling pathway. Thus, we propose that the Rip11/FIP5-kinesin-II complex has a key role in the routing of internalized receptors through the perinuclear recycling endosomes.

The Rab11 subfamily of GTPases plays an important role in vesicle trafficking from endosomes to the plasma membrane. At least six Rab11 effectors (family of Rab11-interacting proteins (FIPs)) have been shown to interact with Rab11 and are hypothesized to regulate various membrane trafficking pathways such as transferrin recycling, cytokinesis, and epidermal growth factor trafficking. In this study, we characterized interactions of FIPs with the Rab11 GTPase using isothermal titration calorimetric studies and mutational analysis. Our data suggest that FIPs cannot differentiate between GTPbound Rab11a and Rab11b in vitro (50 -100 nM affinity) and in vivo. We also show that, although FIPs interact with the GDP-bound form of Rab11 in vitro, the binding affinity (>1000 nM) is not sufficient for FIP and GDPbound Rab11 interactions to occur in vivo. Mutational analysis revealed that both the conserved hydrophobic patch and Tyr 628 are important for the GTP-dependent binding of Rab11 to FIPs. The entropy and enthalpy analyses suggest that binding to Rab11a/b may induce conformational changes in FIPs.

Cytokinesis is a highly regulated and dynamic event that involves the reorganization of the cytoskeleton and membrane compartments. Recently, FIP3 has been implicated in targeting of recycling endosomes to the mid-body of dividing cells and is found required for abscission. Here, we demonstrate that the centralspindlin component Cyk-4 is a FIP3-binding protein. Furthermore, we show that FIP3 binds to Cyk-4 at late telophase and that centralspindlin may be required for FIP3 recruitment to the mid-body. We have mapped the FIP3-binding region on Cyk-4 and show that it overlaps with the ECT2-binding domain. Finally, we demonstrate that FIP3 and ECT2 form mutually exclusive complexes with Cyk-4 and that dissociation of ECT2 from the mid-body at late telophase may be required for the recruitment of FIP3 and recycling endosomes to the cleavage furrow. Thus, we propose that centralspindlin complex not only regulates acto-myosin ring contraction but also endocytic vesicle transport to the cleavage furrow and it does so through sequential interactions with ECT2 and FIP3.

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