Molecular characterization of Rab11-FIP3 binding to ARF GTPases

Abstract

Rab11-FIP3 is a Rab11-binding protein that has been implicated in regulating cytokinesis in mammalian cells. FIP3 functions by simultaneously interacting with Rab11 as well as Arf GTPases. However, unlike the interaction between Rab11 and FIP3, the structural basis of FIP3 binding to Arf GTPases has not yet been determined. The specificity of interaction between FIP3 and Arf GTPases remains controversial. While it was reported that FIP3 preferentially binds to Arf6 some data suggest that FIP3 can also interact with Arf5 and even possibly Arf4. The Arf-interaction motif on FIP3 also remains to be determined. Finally, the importance of Arf binding to FIP3 in regulating cell division and other cellular functions remains unclear. Here we used a combination of various biochemical techniques to measure the affinity of FIP3 binding to various Arfs and to demonstrate that FIP3 predominately interacts with Arf6 in vitro and in vivo. In addition, we identified the motifs mediating Arf6 and FIP3 interaction and demonstrated that FIP3 binds to the Arf6 C-terminus rather than switch motifs. Finally we show that FIP3 and Arf6 binding is required for the targeting of Arf6 to the cleavage furrow during cytokinesis. Thus, we propose that FIP3 is a scaffolding protein that, in addition to regulating endosome targeting to the cleavage furrow, also is required for Arf6 recruitment to the midbody during late telophase.

Introduction

Rab GTPases form the largest family of small monomeric GTPases that play an important role in regulating membrane traffic. Rab proteins cycle between inactive GDP- and active GTP-bound forms. In their active conformation, Rab GTPases regulate the recruitment and/or activation of selective effector proteins such as kinesin and myosin molecular motors, cargo-sorting complexes, lipid kinases and tethering factors.

Rab11a, Rab11b and Rab25 are three closely related Rabs that belong to the Rab11 subfamily (Goldenring et al., 1993, Goldenring et al., 2001). Rab11 GTPases have been shown to regulate several important membrane transport steps that include recycling from endosomes to the plasma membrane, retrograde transport from endosomes to the trans-Golgi network (Wilcke et al., 2000), phagocytosis (Cox et al., 2000), insulin-dependent GLUT4 traffic (Kessler et al., 2000), and cytokinesis (Skop et al., 2001; Pelissier et al., 2003; Riggs et al., 2003; Wilson et al., 2005). In recent years several Rab11 effector proteins have been isolated that include Rab11 family interacting proteins (Rab11-FIPs), also known as FIPs (Prekeris et al., 2000, Prekeris et al., 2001; Hales et al., 2001; Lindsay et al., 2002; Hickson et al., 2003). Based on sequence homology FIPs are classified into two main classes. Class I FIPs (Rip11/FIP5, RCP/FIP1 and FIP2) contain an N-terminal C2 domain. Class II FIPs (FIP3 and FIP4) contain two EF hands (Prekeris, 2003). All FIPs interact with Rab11 GTPases in a GTP-dependent manner with an affinity of 50–200 nM (Junutula et al., 2004; Eathiraj et al., 2006). This interaction is mediated by a highly conserved Rab11 binding domain (RBD) that is situated at the very C-terminus of each FIP (Prekeris et al., 2001; Eathiraj et al., 2006; Jagoe et al., 2006; Shiba et al., 2006). Recently, structures of FIP3/Rab11 and FIP2/Rab11 protein complexes demonstrated that two Rab11 molecules bind to dyad symmetrical sites at the C-terminus of a FIP dimer with a parallel coiled-coil dimer of FIPs (Eathiraj et al., 2006; Jagoe et al., 2006; Shiba et al., 2006).

While class I FIPs have been implicated in regulation of endocytic protein sorting and recycling (Prekeris et al., 2000; Cullis et al., 2002; Hales et al., 2002; Lindsay and McCaffrey, 2002; Peden et al., 2004; Naslavsky et al., 2006), class II FIPs appear to have a more specialized role. In mammalian cells FIP3 and FIP4 have been suggested to regulate endosome targeting to the cleavage furrow during cytokinesis (Horgan et al., 2004; Wilson et al., 2005; Fielding et al., 2005). Similarly, nuclear fallout protein (Nuf), a Drosophila homologue of class II FIPs, was shown to regulate endosomal transport and actin polymerization during cellularization of Drosophila embryos (Riggs et al., 2003). At least to some extent, these specialized functions depend on the ability of FIP3 and FIP4 to bind to Arf GTPases (Fielding et al., 2005). Indeed, FIP3 was originally identified as an Arf5-interacting protein by a yeast two-hybrid screen, and is also sometimes referred to as arfophilin (Shin et al., 1999, Shin et al., 2001). FIP3 and FIP4 have also been shown to interact with Arf6 (Shin et al., 2001; Fielding et al., 2005). Rab11 and Arf GTPases appear to bind to different FIP3 motifs, as FIP3, Rab11 and Arfs can be part of the same protein complex (Fielding et al., 2005).

Unlike the interaction between Rab11 and FIP3, the structural basis of FIP3 binding to Arf GTPases has not yet been determined. The specificity of interaction between FIP3 and Arf GTPases remains controversial. While it was reported that FIP3 preferentially binds to Arf6 (Fielding et al., 2005), some data suggest that FIP3 can also interact with Arf5 and even possibly Arf4 (Shin et al., 1999; Hickson et al., 2003). The Arf-interaction motif on FIP3 also remains to be determined. Finally, the importance of Arf binding to FIP3 in regulating cell division and other cellular functions remains unclear. Here we used a combination of surface plasmon resonance and glutathione bead pull-down assays to measure the affinity of FIP3 binding to various Arfs and localize the Arf-binding site in FIP3. We also used a bacterial two-hybrid system to identify the FIP3-binding region within Arf GTPases. Finally, we used site-directed mutagenesis along with epifluorescence microscopy, FRET and time-lapse microscopy to investigate the functional significance of FIP3 and Arf6 interactions during cytokinesis.