Phosphorylation of Rab11-FIP2 regulates polarity in MDCK cells

The Rab11 effector Rab11-family interacting protein 2 (Rab11-FIP2) regulates transcytosis through its interactions with Rab11a and myosin Vb. Previous studies implicated Rab11-FIP2 in the establishment of polarity in Madin-Darby canine kidney (MDCK) cells through phosphorylation of Ser-227 by MARK2. Here we examine the dynamic role of Rab11-FIP2 phosphorylation on MDCK cell polarity. Endogenous Rab11-FIP2 phosphorylated on Ser-227 coalesces on vesicular plaques during the reestablishment of polarity after either monolayer wounding or calcium switch. Whereas expression of the nonphosphorylatable Rab11-FIP2(S227A) elicits a loss in lumen formation in MDCK cell cysts grown in Matrigel, the putative pseudophosphorylated Rab11-FIP2(S227E) mutant induces the formation of cysts with multiple lumens. On permeable filters, Rab11-FIP2(S227E)-expressing cells exhibit alterations in the composition of both the adherens and tight junctions. At the adherens junction, p120 catenin and K-cadherin are retained, whereas the majority of the E-cadherin is lost. Although ZO-1 is retained at the tight junction, occludin is lost and the claudin composition is altered. Of interest, the effects of Rab11-FIP2 on cellular polarity did not involve myosin Vb or Rab11a. These results indicate that Ser-227 phosphorylation of Rab11-FIP2 regulates the composition of both adherens and tight junctions and is intimately involved in the regulation of polarity in epithelial cells.     

Rab11-FIP2 regulates differentiable steps in transcytosis

Transcytosis through the apical recycling system of polarized cells is regulated by Rab11a and a series of Rab11a-interacting proteins. We have identified a point mutant in Rab11 family interacting protein 2 (Rab11-FIP2) that alters the function of Rab11a-containing trafficking systems. Rab11-FIP2(S229A/R413G) or Rab11-FIP2(R413G) cause the formation of a tubular cisternal structure containing Rab11a and decrease the rate of polymeric IgA transcytosis. The R413G mutation does not alter Rab11-FIP interactions with any known binding partners. Overexpression of Rab11-FIP2(S229A/R413G) alters the localization of a subpopulation of the apical membrane protein GP135. In contrast, Rab11-FIP2(129-512) alters the localization of early endosome protein EEA1. The distributions of both Rab11-FIP2(S229A/R413G) and Rab11-FIP2(129-512) were not dependent on the integrity of the microtubule cytoskeleton. The results indicate that Rab11-FIP2 regulates trafficking at multiple points within the apical recycling system of polarized cells. Rab11a; immunoglobulin A; trafficking; apical recycling; GP135; early endosome; EEA1; Eps15 homology domain     

Rab11 family interacting protein 2 associates with Myosin Vb and regulates plasma membrane recycling

Plasma membrane recycling is an important process necessary for maintaining membrane composition. The motor protein myosin Vb regulates plasma membrane recycling through its association with Rab11a. Overexpression of the tail of myosin Vb disrupts trafficking out of plasma membrane recycling systems and leads to the accumulation of Rab11a in both polarized and non-polarized cells. We have investigated the association of Rab11 family interacting protein 2 (Rab11-FIP2) with myosin Vb as an adapter protein between Rab11a and myosin Vb. Immunofluorescence studies indicated a colocalization of endogenous Rab11-FIP2 with green fluorescent protein-myosin Vb tail overexpressed in Madin-Darby canine kidney (MDCK) cells. Yeast two hybrid assays showed that amino acids 129-356 of Rab11-FIP2 were important for binding to myosin Vb tail. In vitro association assays and co-transfection experiments in both MDCK and HeLa cells confirmed this result but further refined the binding site to amino acids 129-290 of Rab11-FIP2. Like myosin Vb, functional studies indicated that Rab11-FIP2 is also important for normal plasma membrane recycling. Green fluorescent protein-Rab11-FIP2 (129-512), which lacks its amino-terminal C2 domain, functioned as a dominant negative acting truncation that caused accumulation of Rab11a and disrupted IgA trafficking in MDCK cells and transferrin trafficking in HeLa cells. The ternary association of myosin Vb and Rab11-FIP2 with Rab11a suggests that a multimeric protein complex is involved in vesicle trafficking through plasma membrane recycling systems.     

Identification and characterization of a family of Rab11-interacting proteins

Rab11a is a small GTP-binding protein enriched in the pericentriolar plasma membrane recycling systems. We hypothesized that Rab11a-binding proteins exist as downstream effectors of its action. Here we define a family of four Rab11-interacting proteins: Rab11-Family Interacting Protein 1 (Rab11-FIP1), Rab11-Family Interacting Protein 2 (Rab11-FIP2), Rab11-Family Interacting Protein 3 (Rab11-FIP3), and pp75/Rip11. All four interacting proteins associated with wild type Rab11a and dominant active Rab11a (Rab11aS20V) as well as Rab11b and Rab25. Rab11-FIP2 also interacted with dominant negative Rab11a (Rab11aS25N) and the tail of myosin Vb. The binding of Rab11-FIP1, Rab11-FIP2, and Rab11-FIP3 to Rab11a was dependent upon a conserved carboxyl-terminal amphipathic alpha-helix. Rab11-FIP1, Rab11-FIP2, and pp75/Rip11 colocalized with Rab11a in plasma membrane recycling systems in both non-polarized HeLa cells and polarized Madin-Darby canine kidney cells. GFP-Rab11-FIP3 also colocalized with Rab11a in HeLa cells. Rab11-FIP1, Rab11-FIP2, and pp75/Rip11 also coenriched with Rab11a and H(+)K(+)-ATPase on parietal cell tubulovesicles, and Rab11-FIP1 and Rab11-FIP2 translocated with Rab11a and the H(+)K(+)-ATPase upon stimulating parietal cells with histamine. The results suggest that the function of Rab11a in plasma membrane recycling systems is dependent upon a compendium of protein effectors.     

A Role of myosin Vb and Rab11-FIP2 in the aquaporin-2 shuttle

Arginine-vasopressin (AVP) regulates water reabsorption in renal collecting duct principal cells. Its binding to Gs-coupled vasopressin V2 receptors increases cyclic AMP (cAMP) and subsequently elicits the redistribution of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the plasma membrane (AQP2 shuttle), thereby facilitating water reabsorption from primary urine. The AQP2 shuttle is a paradigm for cAMP-dependent exocytic processes. Using sections of rat kidney, the AQP2-expressing cell line CD8, and primary principal cells, we studied the role of the motor protein myosin Vb, its vesicular receptor Rab11, and the myosin Vb- and Rab11-binding protein Rab11-FIP2 in the AQP2 shuttle. Myosin Vb colocalized with AQP2 intracellularly in resting and at the plasma membrane in AVP-treated cells. Rab11 was found on AQP2-bearing vesicles. A dominant-negative myosin Vb tail construct and Rab11-FIP2 lacking the C2 domain (Rab11-FIP2-DeltaC2), which disrupt recycling, caused condensation of AQP2 in a Rab11-positive compartment and abolished the AQP2 shuttle. This effect was dependent on binding of myosin Vb tail and Rab11-FIP2-DeltaC2 to Rab11. In summary, we identified myosin Vb as a motor protein involved in AQP2 recycling and show that myosin Vb- and Rab11-FIP2-dependent recycling of AQP2 is an integral part of the AQP2 shuttle.     

Dynamics of the apical plasma membrane recycling system during cell division

The members of the family of Rab11 small GTPases are critical regulators of the plasma membrane vesicle recycling system. While previous studies have determined that the Golgi apparatus disperses during mitosis and reorganizes after cytokinesis, the fate of the recycling system during the cell cycle is more obscure. We have now studied in MDCK cells the fate during mitosis of an apical recycling system cargo, the polymeric IgA receptor (pIgAR), and regulators of the recycling system, Rab11a and its interacting proteins myosin Vb, Rab11-FIP1, Rab11-FIP2 and pp75/Rip11. Rab11a, pIgAR and myosin Vb containing vesicles dispersed into diffuse puncta in the cytosol during prophase and then became clustered near the spindle poles after metaphase, increasing in intensity throughout telophase. A similar pattern was observed for Rab11-FIP1 and Rab11-FIP2. However, Rab11-FIP1 lost colocalization with other recycling system markers during late prophase, relocating to the pericentriolar material. During telophase, Rab11-FIP1 returned to recycling system vesicles. Western blot analysis indicated that both Rab11a and pIgAR remained associated with membrane vesicles throughout the cell cycle. This behavior of the Rab11a-containing apical recycling endosome system during division was distinct from that of the Golgi apparatus. These results indicate that critical components of the apical recycling system remain associated on vesicles throughout the cell cycle and may provide a means for rapid re-establishment of plasma membrane components after mitosis.     

A Rab11A/myosin Vb/Rab11-FIP2 complex frames two late recycling steps of langerin from the ERC to the plasma membrane

A large body of knowledge relating to the constitution of Rab GTPase/Rab effector complexes and their impact on both membrane domain organization and overall membrane trafficking has been built up in recent years. However in the context of the live cell there are still many questions that remain to be answered, such as where and when these complexes assemble and where they perform their primary function(s). We describe here the dynamic processes that take place in the final steps of the Rab11A dependent recycling pathway, in the context of the membrane platform constituted by Myosin Vb, Rab11A, and Rab11-FIP2. We first confirm that a series of previously reported observations obtained during the study of a number of trafficking cargoes also apply to langerin. Langerin is a cargo molecule that traffics through Rab11A-positive membrane domains of the endosomal recycling pathway. In order to explore the relative dynamics of this set of partners, we make extensive use of a combinatory approach of Live-FRET, fast FRAP video, fast confocal and TIRF microscopy modalities. Our data show that the Myosin Vb/Rab11A/Rab11-FIP2 platform is spatially involved in the regulation of langerin trafficking at two distinct sites within live cells, first at the sorting site in the endosomal recycling compartment (ERC) where transport vesicles are formed, and subsequently, in a strict time-defined order, at the very late stage of docking/tethering and fusion of these langerin recycling vesicles to the plasma membrane.     

Interactions between EHD proteins and Rab11-FIP2: a role for EHD3 in early endosomal transport

Eps15 homology domain (EHD) 1 enables membrane recycling by controlling the exit of internalized molecules from the endocytic recycling compartment (ERC) en route to the plasma membrane, similar to the role described for Rab11. However, no physical or functional connection between Rab11 and EHD-family proteins has been demonstrated yet, and the mode by which they coordinate their regulatory activity remains unknown. Here, we demonstrate that EHD1 and EHD3 (the closest EHD1 paralog), bind to the Rab11-effector Rab11-FIP2 via EH-NPF interactions. The EHD/Rab11-FIP2 associations are affected by the ability of the EHD proteins to bind nucleotides, and Rab11-FIP2 is recruited to EHD-containing membranes. These results are consistent with a coordinated role for EHD1 and Rab11-FIP2 in regulating exit from the ERC. However, because no function has been attributed to EHD3, the significance of its interaction with Rab11-FIP2 remained unclear. Surprisingly, loss of EHD3 expression prevented the delivery of internalized transferrin and early endosomal proteins to the ERC, an effect differing from that described upon EHD1 knockdown. Moreover, the subcellular localization of Rab11-FIP2 and endogenous Rab11 were altered upon EHD3 knockdown, with both proteins absent from the ERC and retained in the cell periphery. The results presented herein promote a coordinated role for EHD proteins and Rab11-FIP2 in mediating endocytic recycling and provide evidence for the function of EHD3 in early endosome to ERC transport.     

Rab11-FIP4 interacts with Rab11 in a GTP-dependent manner and its overexpression condenses the Rab11 positive compartment in HeLa cells

We have recently identified Rab11-FIP4 as the sixth member of the Rab11-FIP family of Rab11 interacting proteins. Here, we demonstrate that Rab11-FIP4 interacts with Rab11 in a GTP-dependent manner and that its C-terminal region allows the protein to self-interact and interact with pp75/Rip11, Rab11-FIP2, and Rab11-FIP3. However, Rab11-FIP4 does not appear to interact directly with Rab coupling protein (RCP). We investigated the subcellular localisation of Rab11-FIP4 in HeLa cells and show that it colocalises extensively with transferrin and with Rab11. Furthermore, when overexpressed, it causes a condensation of the Rab11 compartment in the perinuclear region. We demonstrate that the carboxy-terminal region of Rab11-FIP4 (Rab11-FIP4(C-ter)) is necessary and sufficient for its endosomal membrane association. Expression of Rab11-FIP4(C-ter) causes a dispersal of the Rab11 compartment towards the cell periphery and does not inhibit transferrin recycling in HeLa cells. It is likely that Rab11-FIP4 serves as a Rab11 effector in a Rab11 mediated function other than transferrin recycling.     

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

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.     

Rab11-family interacting protein 2 and myosin Vb are required for CXCR2 recycling and receptor-mediated chemotaxis

Agonist-stimulated internalization followed by recycling to the cell membrane play an important role in fine-tuning the activity of chemokine receptors. Because the recycling of chemokine receptors is critical for the reestablishment of the cellular responsiveness to ligand, it is crucial to understand the mechanisms underlying the receptor recycling and resensitization. In the present study, we have demonstrated that the chemokine receptor CXCR2 associated with myosin Vb and Rab11-family interacting protein 2 (FIP2) in a ligand-dependent manner. Truncation of the C-terminal domain of the receptor did not affect the association, suggesting that the interactions occur upstream of the C terminus of CXCR2. After ligand stimulation, the internalized CXCR2 colocalized with myosin Vb and Rab11-FIP2 in Rab11a-positive vesicles. The colocalization lasted for approximately 2 h, and little colocalization was observed after 4 h of ligand stimulation. CXCR2 also colocalized with myosin Vb tail or Rab11-FIP2 (129-512), the N-terminal-truncated mutants of myosin Vb and Rab11-FIP2, respectively, but in a highly condensed manner. Expression of the enhanced green fluorescent protein-tagged myosin Vb tail significantly retarded the recycling and resensitization of CXCR2. CXCR2 recycling was also reduced by the expression Rab11-FIP2 (129-512). Moreover, expression of the myosin Vb tail reduced CXCR2- and CXCR4-mediated chemotaxis. These data indicate that Rab11-FIP2 and myosin Vb regulate CXCR2 recycling and receptor-mediated chemotaxis and that passage of internalized CXCR2 through Rab11a-positive recycling system is critical for physiological response to a chemokine.     

Rab11-FIP3 is critical for the structural integrity of the endosomal recycling compartment

Rab11-FIP3 is an endosomal recycling compartment (ERC) protein that is implicated in the process of membrane delivery from the ERC to sites of membrane insertion during cell division. Here we report that Rab11-FIP3 is critical for the structural integrity of the ERC during interphase. We demonstrate that knockdown of Rab11-FIP3 and expression of a mutant of Rab11-FIP3 that is Rab11-binding deficient cause loss of all ERC-marker protein staining from the pericentrosomal region of A431 cells. Furthermore, we find that fluorophore-labelled transferrin cannot access the pericentrosomal region of cells in which Rab11-FIP3 function has been perturbed. We find that this Rab11-FIP3 function appears to be specific because expression of the equivalent Rab11-binding deficient mutant of Rab-coupling protein does not perturb ERC morphology. In addition, we find that other organelles such as sorting and late endosomes are unaffected by loss of Rab11-FIP3 function. Finally, we demonstrate the presence of an extensive coiled-coil region between residues 463 and 692 of Rab11-FIP3, which exists as a dimer in solution and is critical to support its function on the ERC. Together, these data indicate that Rab11-FIP3 is necessary for the structural integrity of the pericentrosomal ERC.     

Rab11-FIP2 suppressed tumor growth via regulation of PGK1 ubiquitination in non-small cell lung cancer

Mounting evidence has shown that the Rab11-FIP2 has critical roles in cancer cell growth. However, the clinical significance of Rab11-FIP2 in Non-small cell lung cancer (NSCLC) remains to be fully elucidated. In this study, we investigated the expression of Rab11-FIP2 using immunohistochemistry in 150 patients with NSCLC. We found that its expression level in NSCLC was much lower than that in the corresponding adjacent normal tissues. The DNA methylation data revealed that Rab11-FIP2 were significantly hypermethylated in NSCLC. The methylation level in the gene body was negatively correlated with the expression level of Rab11-FIP2 in NSCLC. Furthermore, enforced expression of Rab11-FIP2 dramatically reduced cancer cell proliferation and tumorigenesis, indicating a tumor suppressor role of PGK1 in NSCLC progression. Mechanistic investigations showed that Rab11-FIP2 interacted with the glycolytic kinase PGK1 and promoted its ubiquitination in NSCLC cells, leading to inactivation of the oncogenic AKT/mTOR signaling pathway. Overall, our data indicate that reduced expression of Rab11-FIP2 by DNA hypermethylation plays an important role in NSCLC tumor growth.     

A kinase cascade leading to Rab11-FIP5 controls transcytosis of the polymeric immunoglobulin receptor

Polymeric immunoglobulin A (pIgA) transcytosis, mediated by the polymeric immunoglobulin receptor (pIgR), is a central component of mucosal immunity and a model for regulation of polarized epithelial membrane traffic. Binding of pIgA to pIgR stimulates transcytosis in a process requiring Yes, a Src family tyrosine kinase (SFK). We show that Yes directly phosphorylates EGF receptor (EGFR) on liver endosomes. Injection of pIgA into rats induced EGFR phosphorylation. Similarly, in MDCK cells, pIgA treatment significantly increased phosphorylation of EGFR on various sites, subsequently activating extracellular signal-regulated protein kinase (ERK). Furthermore, we find that the Rab11 effector Rab11-FIP5 is a substrate of ERK. Knocking down Yes or Rab11-FIP5, or inhibition of the Yes-EGFR-ERK cascade, decreased pIgA-pIgR transcytosis. Finally, we demonstrate that Rab11-FIP5 phosphorylation by ERK controls Rab11a endosome distribution and pIgA-pIgR transcytosis. Our results reveal a novel Yes-EGFR-ERK-FIP5 signalling network for regulation of pIgA-pIgR transcytosis.     

A Highlights from MBoC Selection: Rab11-family interacting proteins define spatially and temporally distinct regions within the dynamic Rab11a-dependent recycling system

The Rab11-family interacting proteins (Rab11-FIPs) facilitate Rab11-dependent vesicle recycling. We hypothesized that Rab11-FIPs define discrete subdomains and carry out temporally distinct roles within the recycling system. We used live-cell deconvolution microscopy of HeLa cells expressing chimeric fluorescent Rab11-FIPs to examine Rab11-FIP localization, transferrin passage through Rab11-FIP–containing compartments, and overlap among Rab11-FIPs within the recycling system. FIP1A, FIP2, and FIP5 occupy widely distributed mobile tubules and vesicles, whereas FIP1B, FIP1C, and FIP3 localize to perinuclear tubules. Internalized transferrin entered Rab11-FIP–containing compartments within 5 min, reaching maximum colocalization with FIP1B and FIP2 early in the time course, whereas localization with FIP1A, FIP1C, FIP3, and FIP5 was delayed until 10 min or later. Whereas direct interactions with FIP1A were only observed for FIP1B and FIP1C, FIP1A also associated with membranes containing FIP3. Live-cell dual-expression studies of Rab11-FIPs revealed the tubular dynamics of Rab11-FIP–containing compartments and demonstrated a series of selective associations among Rab11-FIPs in real time. These findings suggest that Rab11-FIP1 proteins participate in spatially and temporally distinct steps of the recycling process along a complex and dynamic tubular network in which Rab11-FIPs occupy discrete domains.     

Differential, phosphorylation dependent trafficking of AQP2 in LLC-PK1 cells

The kidney maintains water homeostasis by modulating aquaporin 2 (AQP2) on the plasma membrane of collecting duct principal cells in response to vasopressin (VP). VP mediated phosphorylation of AQP2 at serine 256 is critical for this effect. However, the role of phosphorylation of other serine residues in the AQP2 C-terminus is less well understood. Here, we examined the effect of phosphorylation of S256, S261 and S269 on AQP2 trafficking and association with recycling pathway markers. We used LLC-PK1 cells expressing AQP2(S-D) or (S-A) phospho mutants and a 20°C cold block, which allows endocytosis to continue, but prevents protein exit from the trans Golgi network (TGN), inducing formation of a perinuclear AQP2 patch. AQP2-S256D persists on the plasma membrane during cold block, while wild type AQP2, AQP2-S256A, S261A, S269A and S269D are internalized and accumulate in the patch. Development of this patch, a measure of AQP2 internalization, was most rapid with AQP2-S256A, and slowest with S261A and S269D. AQP2-S269D exhibited a biphasic internalization profile with a significant amount not internalized until 150 minutes of cold block. After rewarming to 37°C, wt AQP2, AQP2-S261A and AQP2-S269D rapidly redistributed throughout the cytoplasm within 20 minutes, whereas AQP2-S256A dissipated more slowly. Colocalization of AQP2 mutants with several key vesicular markers including clathrin, HSP70/HSC70, EEA, GM130 and Rab11 revealed no major differences. Overall, our data provide evidence supporting the role of S256 and S269 in the maintenance of AQP2 at the cell surface and reveal the dynamics of internalization and recycling of differentially phosphorylated AQP2 in cell culture.     

Rab11-FIP2, an adaptor protein connecting cellular components involved in internalization and recycling of epidermal growth factor receptors

Rab11-FIP2 is a member of a newly identified family of Rab11-binding proteins that have been implicated in the function of recycling endosomes. Here we show that Rab11-FIP2 may also be involved with the process of receptor-mediated endocytosis. First we demonstrate that Rab11-FIP2 contains an NPF motif that allows it to bind Reps1, a member of a family of EH domain proteins involved in endocytosis. We also show that Rab11-FIP2 associates with the alpha-adaptin subunit of AP-2 complexes, which are known to recruit receptors into clathrin-coated vesicles. Finally, we find that overexpression of Rab11-FIP2 suppresses the internalization of epidermal growth factor receptors, but not transferrin receptors, through binding sites that promote complex formation with Rab11, Reps1, and alpha-adaptin. These findings suggest that Rab11-FIP2 may participate in the coupling of receptor-mediated endocytosis to the subsequent sorting of receptor-containing vesicles in endosomes.     

Rab11-FIP2 functions in transferrin recycling and associates with endosomal membranes via its COOH-terminal domain

Rab11-FIP2 is a recently described member of the Rip11/Rab11-FIP/Rab coupling protein family of Rab11 interacting proteins. Rab11-FIP2 interacts with both Rab11 and myosin Vb and co-localizes with Rab11 in both HeLa and Madin-Darby canine kidney cells (Hales, C. M., Griner, R., Hobdy-Henderson, K. C., Dorn, M. C., Hardy, D., Kumar, R., Navarre, J., Chan, E. K., Lapierre, L. A., and Goldenring, J. R. (2001) J. Biol. Chem. 276, 39067-390751). Here, we characterized the specificity of the interaction between Rab11-FIP2 and Rab11 and report that it does not interact with Rab4, Rab3, Rab5, Rab6, or Rab7. We demonstrate that the COOH-terminal region of Rab11-FIP2, which contains the Rab11 binding domain (RBD), is necessary and sufficient for its early endosomal membrane association. In contrast, the amino-terminal region, which contains a phospholipid binding C2-domain, by itself was insufficient for membrane binding. Expression of a deletion mutant of Rab11-FIP2, containing the RBD, caused tubulation of a transferrin receptor-positive early endosomal compartment in HeLa cells. Endogenous Rab11 was also associated with this compartment. This phenotype cannot be reversed by excess wild-type Rab11, or dominant-positive Rab11 (Rab11Q70L), suggesting that Rab11-FIP2 functions downstream of Rab11 in endosomal trafficking.