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The C2 Domains of the Class I Rab11 Family of Interacting Proteins Target Recycling Vesicles to the Plasma Membrane

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The C2 Domains of the Class I Rab11 Family of Interacting Proteins Target Recycling Vesicles to the Plasma Membrane Research Article 4365 The C2 domains of the class I Rab11 family of interacting proteins target recycling vesicles to the plasma membrane Andrew J. Lindsay and Mary W. McCaffrey* Molecular Cell Biology Laboratory, Department of Biochemistry, Biosciences Institute, University College Cork, Cork, Ireland *Author for correspondence (e-mail: [email protected]) Accepted 21 April 2004 Journal of Cell Science 117, 4365-4375 Published by The Company of Biologists 2004 doi:10.1242/jcs.01280 Summary The Rab11 family of interacting proteins (Rab11-FIP) is a phosphatidylinositol-(3,4,5)-trisphosphate [PtdIns(3,4,5)P3] recently identified protein family composed of, to date, six and the second messenger phosphatidic acid. Stimulation of members that interact with Rab11. They all share a highly PtdIns(3,4,5)P3 or phosphatidic acid synthesis results in the homologous Rab11-binding domain (RBD) at their C- translocation of the Rab11-FIPs from a perinuclear location termini. However, apart from the RBD, they vary in their to the periphery of the cell. By contrast, the transferrin domain organization. Rab11-FIP3 and Rab11-FIP4 possess receptor does not translocate to the plasma membrane an ezrin-radixin-moesin (ERM) domain in their C-terminal under these conditions. This translocation is dependent on half and EF hands in their N-terminal region. They have the presence of the C2 domain, because class I Rab11-FIP been termed class II Rab11-FIPs. The class I Rab11-FIPs, green-fluorescent-protein fusions that lack the C2 domain Rab coupling protein (RCP), Rip11 and Rab11-FIP2, each cannot translocate to the plasma membrane. We propose have a C2 phospholipid-binding domain near their N- that the C2 domains of the class I Rab11-FIPs function to termini. Although they are still membrane associated, target these proteins to ‘docking sites’ in the plasma truncation mutants of the class I Rab11-FIPs that lack their membrane that are enriched in PtdIns(3,4,5)P3 and C2 domains display an altered subcellular distribution in phosphatidic acid. vivo, indicating that this domain plays an important role in specifying their correct intracellular localization. To determine the phospholipids to which they bind, a protein Key words: Rab11-FIPs, C2 domain, phosphatidylinositol (3,4,5)- phospholipid overlay assay was performed. Our results trisphosphate, endocytic recycling compartment, Phosphatidic acid, indicate that the class-I Rab11-FIPs bind preferentially to Phospholipase D Introduction terminus. They are predominantly membrane bound and Rab11 is a small GTPase that plays a role in regulating localized to the ERC under steady-state conditions. membrane traffic from the endocytic recycling compartment C2 domains are found in many proteins that are involved in (ERC) to either the plasma membrane or the trans-Golgi cell signalling [e.g. phosphoinositide-3-kinase (PI-3-kinase) network (TGN). Recently, several Rab11-interacting proteins and PTEN] and membrane trafficking (e.g. rabphilin-3A and have been identified that share a highly homologous Rab11- synaptotagmin). They are approximately 130 amino acid binding domain (RBD) (Hales et al., 2001; Lindsay et al., motifs, first identified in protein kinase C (PKC) (Cho, 2001), 2002; Prekeris et al., 2000). Owing to the presence of this which serve as protein-phospholipid and protein-protein RBD, these proteins were grouped into the same family, binding modules. Their phospholipid binding can be Ca2+ termed the Rab11 family of interacting proteins (Rab11-FIP). dependent or Ca2+ independent. In some cases, the C2 domain However, apart from the RBD, they have distinct domain is involved in the translocation of the protein to specific regions organizations, allowing them to be subdivided into two within the cell. For example, the C2 domains of conventional classes. The class II Rab11-FIPs include Rab11-FIP3 and PKCs, which bind anionic phospholipids, mediate the rapid Rab11-FIP4, and possess an ezrin-radixin-moesin (ERM) translocation of PKC to the plasma membrane (Oancea and domain, EF hands and a proline-rich domain, and have been Meyer, 1998). By contrast, the C2 domain of cytosolic localized to the ERC, TGN and centrosome (Hales et al., phospholipase A2 (cPLA2) translocates to the nuclear envelope 2001; Hickson et al., 2003; Wallace et al., 2002). Rab11-FIP3 upon Ca2+ influx (Gijon et al., 1999; Perisic et al., 1999). and Rab11-FIP4 were also independently identified as Arf- Several studies have demonstrated that expression of GTPase-binding proteins and termed arfophilin 1 and truncation mutants of the Rab11-FIPs that lack their C2 arfophilin 2 (Hickson et al., 2003; Shin et al., 2001; Shin et domains results in a significant inhibition in the rate of al., 1999). Rab coupling protein (RCP), Rip11 and Rab11- endosomal recycling (Lindsay et al., 2002; Lindsay and FIP2, comprise the class I Rab11-FIPs. In addition to the C- McCaffrey, 2002; Prekeris et al., 2000). Transferrin is retained terminal RBD, they also possess a C2 domain near their N- in an intricate tubular network within the cell and its recycling 4366 Journal of Cell Science 117 (19) back to the plasma membrane is inhibited. This indicates that Plasmid constructs the C2 domain of the class I Rab11-FIPs is likely to play an Fusions between green fluorescent protein (GFP) and RCP and important role in regulating the transport of ligands and their Rab11-FIP2 have been described previously (Lindsay et al., 2002; receptors back to the plasma membrane. We show here that the Lindsay and McCaffrey, 2002). GFP-Rip11 was generated by in vivo localization of the C2 domain truncation mutants of the polymerase chain reaction (PCR) using pBluescriptIISK KIAA0857 class I Rab11-FIPs, while membrane associated, is different (Kazusa DNA Research Institute) as template and the primers Rip11FWD (5′-CGGAATTCGCCCTGCGGGGCGCGGAG-3′) and from that of the wild-type proteins. ′ We have previously demonstrated that the C-terminal 65 Rip11REV (5 -CGGAATTCGCACAGAACCTGATGCCTCCAAG- 3′). The product was digested and ligated into the EcoRI site of amino acids of RCP and Rab11-FIP2, which encompass their α pEGFP-C1 (Clontech). GFP-RCP(1-199) was generated by ligating the -helical coil and RBD, are sufficient for membrane binding ~600 bp SalI fragment from pEGFP-C3 RCP into the SalI site of (Lindsay et al., 2002; Lindsay and McCaffrey, 2002). This pEGFP-C3. GFP-RCP(200-649) was constructed by ligating the SalI- suggests that the C2 domain is not required for the recruitment BamHI fragment from pEGFP-C3 RCP into the SalI-BamHI site of of the Rab11-FIPs from the cytosol to intracellular membranes. pEGFP-C1. GFP-Rab11-FIP2(1-284) was generated by PCR using Given this observation, there are several possible roles that the Rab11-FIP2FWD (5′-CGGAATTCCCTGTCCGAGCAAGCCCAA- C2 domains of the class I Rab11-FIPs might play. One AAG-3′) and Rab11-FIP2∆C2REV (5′-CGGAATTCGAAACCAG- hypothesis is that the three C2 domains bind different CCACAGGATCAATTC-3′) and ligating into the EcoRI site of phosholipid(s) and thus provide a means by which each Rab11- pEGFP-C1. GFP-Rab11-FIP2(277-512) was generated by ligating the FIP can sense a specific membrane microdomain and thus HindIII-BamHI fragment from pEGFP-C1 Rab11-FIP2 into the HindIII-BamHI site of pEGFP-C2 (Clontech). GFP-Rip11(1-218) was provide a mechanism for ‘fine-tuning’ their subcellular generated by digesting pEGFP-C1 Rip11 with BamHI and religating localization. Data from the Scheller group revealed that the backbone. GFP-Rip11(219-653) was constructed by ligating the ~2.2 treatment of MDCK cells with kinase inhibitors results in the kb BamHI fragment from pBSKII KIAA0857 into pEGFP-C1. RCP(1- translocation of Rip11 from the ERC to the plasma membrane. 199) was subcloned into pGEX2T (Amersham Biosciences). Rip11(1- Thus, it was proposed that Rip11 traffics between these two 218) was subcloned into the BamHI site of pGEX3X and Rab11- locations (Prekeris et al., 2000). It is therefore possible that the FIP2(1-284) was subcloned into the EcoRI site of pGEX3X (Amersham C2 domains function to target vesicles carrying the Rab11-FIPs Biosciences). All constructs generated by PCR were confirmed by to the plasma membrane. A third possibility is that the C2 sequencing. domains function as protein-protein interaction modules, in a mode similar to synaptotagmin, which binds syntaxin at the Expression and purification of GST fusion proteins plasma membrane in a calcium-dependent manner (Katan and Glutathione-S-transferase (GST) fusion-protein expression was Allen, 1999). induced in E. coli XL-1 Blue cells with 0.1 mM isopropyl- With these possibilities in mind, we used a protein- thiogalactoside (IPTG) at 30°C for 4 hours. The bacterial cells were phospholipid overlay technique and determined that all three resuspended in 1× PBS and lysed with 1% Triton X-100 plus 5 mM C2 domains bind preferentially to PtdIns(3,4,5)P3 and dithiothreitol. The lysate was clarified by centrifugation at 12,000 g phosphatidic acid (PA). We found that synthesis of either and the protein was bound to glutathione-agarose (Sigma), which was PtdIns(3,4,5)P3 or PA by stimulating A431 cells with washed twice with PBS. The protein was eluted with 10 mM epidermal growth factor (EGF) or a phorbol ester resulted in glutathione and subsequently dialysed overnight in PBS. the translocation of endogenous RCP and Rab11-FIP2 from the ERC to the plasma membrane. Treatment of cells with Protein-phospholipid assays wortmannin before stimulation abolished this translocation. The protein-phospholipid overlay assays were carried out according Plasma membrane translocation was dependent on the to the manufacturers instructions. Briefly, PipArrays (Echelon presence of the C2 domain because truncation mutants Biosciences) were blocked in 3% bovine serum albumin (BSA) in lacking this domain did not translocate in treated cells.
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