GRAB is a binding partner for the Rab11a and Rab11b GTPases

Co-ordination of Rab GTPase function has emerged as a crucial mechanism in the control of intracellular trafficking processes in eukaryotic cells. Here, we show that GRAB/Rab3IL1 [guanine nucleotide exchange factor for Rab3A; RAB3A interacting protein (rabin3)-like 1], a protein that has previously be shown to act as a GEF (guanine nucleotide exchange factor) for Rab3a, Rab8a and Rab8b, is also a binding partner for Rab11a and Rab11b, but not the closely related Rab25 GTPase. We demonstrate that exogenous expression of Rab11a and Rab11b shift GRAB’s distribution from the cytoplasm onto membranes. We find that the Rab11a/Rab11b-binding region of GRAB lies within its carboxy-terminus, a region distinct from its GEF domain and Rab3a-binding region. Finally, we describe a GRAB deletion mutant (GRAB_Δ223–228) that is deficient in Rab11-binding ability. These data identify GRAB as a dual Rab-binding protein that could potentially link Rab3 and Rab11 and/or Rab8 and Rab11-mediated intracellular trafficking processes.     

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.     

Munc13-4 Is a Rab11-binding Protein That Regulates Rab11-positive Vesicle Trafficking and Docking at the Plasma Membrane

The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane.     

Trafficking of cGMP-dependent protein kinase II via interaction with Rab11

cGMP-dependent protein kinase II (cGK-II) is implicated in several physiological functions including intestinal secretion, bone growth, and learning and memory, but the detailed mechanisms are still unclear. To identify proteins that are involved in cGMP/cGK-II signaling, we performed yeast two-hybrid screening and identified Rab11b as a cGK-II-interacting protein that regulates the slow-recycling pathway. Interestingly, cGK-II interacted with the GDP-bound form of Rab11b (Rab11b S25N), but not the GTP-bound form, in mammalian cells. Immunofluorescence staining revealed that Rab11b S25N promoted the translocation of cGK-II from the plasma membrane to the cytoplasm and that the localization of cGK-II extensively overlapped with Rab11b. Furthermore, treatment with a membrane-permeable cGMP analog caused the rapid retranslocation of cGK-II and Rab11b S25N to the membrane. These data indicate that Rab11b is necessary for the trafficking of cGK-II and that the cGMP/cGK-II signaling pathway is closely related to Rab11b recycling pathway.     

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‐FIP2 Interaction with MYO5B Regulates Movement of Rab11a‐Containing Recycling Vesicles

A tripartite association of Rab11a with both Rab11‐FIP2 and MYO5B regulates recycling endosome trafficking. We sought to define the intermolecular interactions required between Rab11‐FIP2 and MYO5B. Using a random mutagenesis strategy, we identified point mutations at S229P or G233E in Rab11‐FIP2 that caused loss of interaction with MYO5B in yeast two‐hybrid assays as well as loss of interaction of Rab11‐FIP2(129‐356) with MYO5B tail when expressed in HeLa cells. Single mutations or the double S229P/G233E mutation failed to alter the association of full‐length Rab11‐FIP2 with MYO5B tail in HeLa cells. While EGFP‐Rab11‐FIP2 wild type colocalized with endogenous MYO5B staining in MDCK cells, EGFP‐Rab11‐FIP2(S229P/G233E) showed a significant decrease in localization with endogenous MYO5B. Analysis of Rab11a‐containing vesicle movement in live HeLa cells demonstrated that when the MYO5B/Rab11‐FIP2 association is perturbed by mutation or by Rab11‐FIP2 knockdown, vesicle movement is increased in both speed and track length, consistent with an impairment of MYO5B tethering at the cytoskeleton. These results support a critical role for the interaction of MYO5B with Rab11‐FIP2 in stabilizing the functional complex with Rab11a, which regulates dynamic movements of membrane recycling vesicles.      

Recycling of the human prostacyclin receptor is regulated through a direct interaction with Rab11a GTPase

The human prostacyclin receptor (hIP) undergoes agonist-induced internalization but the mechanisms regulating its intracellular trafficking and/or recycling to the plasma membrane are poorly understood. Herein, we conducted a yeast-two-hybrid screen to identify proteins interacting with the carboxyl-terminal (C)-tail domain of the hIP and discovered a novel interaction with Rab11a. This interaction was confirmed by co-immunoprecipitations in mammalian HEK293 and was augmented by cicaprost stimulation. The hIP co-localized to Rab11-containing recycling endosomes in both HEK293 and endothelial EA.hy 926 cells in a time-dependent manner following cicaprost stimulation. Moreover, over-expression of Rab11a significantly increased recycling of the hIP, while the dominant negative Rab11^S25N impaired that recycling. Conversely, while the hIP co-localized to Rab4-positive endosomes in response to cicaprost, ectopic expression of Rab4a did not substantially affect overall recycling nor did Rab4a directly interact with the hIP. The specific interaction between the hIP and Rab11a was dependent on a 22 amino acid (Val²⁹⁹–Gln³²⁰) sequence within its C-tail domain and was independent of isoprenylation of the hIP. This study elucidates a critical role for Rab11a in regulating trafficking of the hIP and has identified a novel Rab11 binding domain (RBD) within its C-tail domain that is both necessary and sufficient to mediate interaction with Rab11a.     

Rab11b Regulates the Apical Recycling of the Cystic Fibrosis Transmembrane Conductance Regulator in Polarized Intestinal Epithelial Cells

The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP/PKA-activated anion channel, undergoes efficient apical recycling in polarized epithelia. The regulatory mechanisms underlying CFTR recycling are understood poorly, yet this process is required for proper channel copy number at the apical membrane, and it is defective in the common CFTR mutant, ΔF508. Herein, we investigated the function of Rab11 isoforms in regulating CFTR trafficking in T84 cells, a colonic epithelial line that expresses CFTR endogenously. Western blotting of immunoisolated Rab11a or Rab11b vesicles revealed localization of endogenous CFTR within both compartments. CFTR function assays performed on T84 cells expressing the Rab11a or Rab11b GDP-locked S25N mutants demonstrated that only the Rab11b mutant inhibited 80% of the cAMP-activated halide efflux and that only the constitutively active Rab11b-Q70L increased the rate constant for stimulated halide efflux. Similarly, RNAi knockdown of Rab11b, but not Rab11a, reduced by 50% the CFTR-mediated anion conductance response. In polarized T84 monolayers, adenoviral expression of Rab11b-S25N resulted in a 70% inhibition of forskolin-stimulated transepithelial anion secretion and a 50% decrease in apical membrane CFTR as assessed by cell surface biotinylation. Biotin protection assays revealed a robust inhibition of CFTR recycling in polarized T84 cells expressing Rab11b-S25N, demonstrating the selective requirement for the Rab11b isoform. This is the first report detailing apical CFTR recycling in a native expression system and to demonstrate that Rab11b regulates apical recycling in polarized epithelial cells.     

Rab11 is essential for fertility in Drosophila

Rab11, a small GTP binding protein involved in vesicular trafficking, has emerged as a key player in regulating various cellular events during Drosophila development and differentiation. In our earlier study a P-insertion line, Rab11(mo), was established as a new hypomorphic allele of Rab11 gene, showing degenerated eye phenotype, bristle abnormalities and sterility. We show here that Rab11 is expressed in the entire testis, more prominently in the secretory cells, and in ovary it is localized at the posterior pole. Rab11(mo) males and females are sterile. The sterility in males has been attributed to defects in the sperm individualization process, while in females, cytoskeleton disruption and reduction/loss of the posteriorly localized protein, Vasa, as a consequence of loss/mislocalization of Rab11 might be the cause of sterility. Fertility as well as the posterior localization of Rab11 and Vasa or cytoskeleton integrity was restored in pCaSpeR4-Rab11/+; Rab11(mo)/Rab11(mo) egg chambers, confirming the requirement of Rab11 in these events.     

Regulation of vesicle trafficking in madin-darby canine kidney cells by Rab11a and Rab25

Polarized epithelial cells maintain the polarized distribution of basolateral and apical membrane proteins through a process of receptor-mediated endocytosis, sorting, and then recycling to the appropriate membrane domain. We have previously shown that the small GTP-binding proteins, Rab11a and Rab25, are associated with the apical recycling system of Madin-Darby canine kidney cells. Here we have utilized inducible expression of wild-type, dominant negative, and constitutively active mutants to directly compare the functions of Rab25 and Rab11a in postendocytic vesicular transport. We found that a Rab11a mutant deficient in GTP binding, Rab11aS25N, potently inhibited both transcytosis and apical recycling yet failed to inhibit transferrin recycling. Similarly, expression of either wild type Rab25 or the active mutant Rab25S21V inhibited both apical recycling and transcytosis of IgA by greater than 50% but had no effect on basolateral recycling of transferrin. Interestingly, the GTPase-deficient mutant Rab11aS20V inhibited basolateral to apical transcytosis of IgA, but had no effect on either apical or basolateral recycling. These results indicate that neither Rab11a nor Rab25 function in the basolateral recycling of transferrin in polarized Madin-Darby canine kidney cells cells, consistent with recent morphological observations by others. Thus, transferrin receptors must be recycled to the plasma membrane prior to sorting of apically directed cargoes into Rab11a/Rab25-positive apical recycling endosomes.     

Rab11 Regulates the Mast Cell Exocytic Response

Stimulated exocytic events provide a means for physiological communication and are a hallmark of the mast cell‐mediated allergic response. In mast cells these processes are triggered by antigen crosslinking of IgE bound to its high‐affinity receptor, Fc?RI, on the cell surface. Here we use the endosomal v‐SNARE VAMP8, and the lysosomal hydrolase β‐hexosaminidase (β‐Hex), each C‐terminally fused to super‐ecliptic pHluorin, to monitor stimulated exocytosis. Using these pHluorin‐tagged constructs, we monitor stimulated exocytosis by fluorimetry and visualize individual exocytic events with total internal reflection (TIRF) microscopy. Similar to constitutive recycling endosome (RE) trafficking, we find that stimulated RE exocytosis, monitored by VAMP8, is attenuated by expression of dominant negative (S25N) Rab11. Stimulated β‐Hex exocytosis is also reduced in the presence of S25N Rab11, suggesting that expression of this mutant broadly impacts exocytosis. Interestingly, pretreatment with inhibitors of actin polymerization, cytochalasin D or latrunculin A, substantially restores both RE and lysosome exocytosis in cells expressing S25N Rab11. Conversely, stabilizing F‐actin with jasplakinolide inhibits antigen‐stimulated exocytosis but is not additive with S25N Rab11‐mediated inhibition, suggesting that these reagents inhibit related processes. Together, our results suggest that Rab11 participates in the regulation necessary for depolymerization of the actin cytoskeleton during stimulated exocytosis in mast cells.      

Global Ablation of the Mouse Rab11a Gene Impairs Early Embryogenesis and Matrix Metalloproteinase Secretion

Rab11a has been conceived as a prominent regulatory component of the recycling endosome, which acts as a nexus in the endo- and exocytotic networks. The precise in vivo role of Rab11a in mouse embryonic development is unknown. We globally ablated Rab11a and examined the phenotypic and molecular outcomes in Rab11a^null blastocysts and mouse embryonic fibroblasts. Using multiple trafficking assays and complementation analyses, we determined, among multiple important membrane-associated and soluble cargos, the critical contribution of Rab11a vesicular traffic to the secretion of multiple soluble MMPs. Rab11a^null embryos were able to properly form normal blastocysts but died at peri-implantation stages. Our data suggest that Rab11a critically controls mouse blastocyst development and soluble matrix metalloproteinase secretion.     

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.     

Association of Rab25 and Rab11a with the Apical Recycling System of Polarized Madin–Darby Canine Kidney Cells

Recent evidence suggests that apical and basolateral endocytic pathways in epithelia converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. We report here that in the epithelial cell line Madin–Darby Canine Kidney (MDCK), antibodies to Rab11a label an apical pericentriolar endosomal compartment that is dependent on intact microtubules for its integrity. Furthermore, this compartment is accessible to a membrane-bound marker (dimeric immunoglobulin A [IgA]) internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome. We have also examined the role of a closely related epithelial-specific Rab, Rab25, in the regulation of membrane recycling and transcytosis in MDCK cells. When cDNA encoding Rab25 was transfected into MDCK cells, the protein colocalized with Rab11a in subapical vesicles. Rab25 transfection also altered the distribution of Rab11a, causing the coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways.     

Small GTPase Rab11b regulates degradation of surface membrane L-type Cav1.2 channels

L-type Ca(2+) channels (LTCCs) play a critical role in Ca(2+)-dependent signaling processes in a variety of cell types. The number of functional LTCCs at the plasma membrane strongly influences the strength and duration of Ca(2+) signals. Recent studies demonstrated that endosomal trafficking provides a mechanism for dynamic changes in LTCC surface membrane density. The purpose of the current study was to determine whether the small GTPase Rab11b, a known regulator of endosomal recycling, impacts plasmalemmal expression of Ca(v)1.2 LTCCs. Disruption of endogenous Rab11b function with a dominant negative Rab11b S25N mutant led to a significant 64% increase in peak L-type Ba(2+) current (I(Ba,L)) in human embryonic kidney (HEK)293 cells. Short-hairpin RNA (shRNA)-mediated knockdown of Rab11b also significantly increased peak I(Ba,L) by 66% compared when with cells transfected with control shRNA, whereas knockdown of Rab11a did not impact I(Ba,L). Rab11b S25N led to a 1.7-fold increase in plasma membrane density of hemagglutinin epitope-tagged Ca(v)1.2 expressed in HEK293 cells. Cell surface biotinylation experiments demonstrated that Rab11b S25N does not significantly impact anterograde trafficking of LTCCs to the surface membrane but rather slows degradation of plasmalemmal Ca(v)1.2 channels. We further demonstrated Rab11b expression in ventricular myocardium and showed that Rab11b S25N significantly increases peak I(Ba,L) by 98% in neonatal mouse cardiac myocytes. These findings reveal a novel role for Rab11b in limiting, rather than promoting, the plasma membrane expression of Ca(v)1.2 LTCCs in contrast to its effects on other ion channels including human ether-a-go-go-related gene (hERG) K(+) channels and cystic fibrosis transmembrane conductance regulator. This suggests Rab11b differentially regulates the trafficking of distinct cargo and extends our understanding of how endosomal transport impacts the functional expression of LTCCs.     

Rab11 is required for tubulogenesis of Malpighian tubules in Drosophila melanogaster

Intracellular vesicular trafficking is one of the important tools in maintaining polarity, adhesion, and shape of epithelial cells. Rab11, a subfamily of the Ypt/Rab gene family of ubiquitously expressed GTPases and a molecular marker of recycling endosomes, transports different components of plasma membrane. Here, we report that Rab11 affects tubulogenesis of Malpighian tubules (MTs). MTs are simple polarized epithelial tubular structures, considered as functional analogue of human kidney. Rab11 has pleiotropic effects on MTs development as down‐regulation of Rab11 in principal cells (PCs) of MTs from embryonic stages of development results in reduced endoreplication, clustering of cells, disorganized cytoskeleton, and disruption of polarity leading to shortening of MTs in third instar larvae. Rab11 is also required for proper localization of different transporters in PCs, essential for physiological activity of MTs. Collectively, our data suggest that Rab11 plays a key role in the process of tubulogenesis of MTs in Drosophila.     

The crystal structure of the small GTPase Rab11b reveals critical differences relative to the Rab11a isoform

Rab GTPases constitute the largest family of small monomeric GTPases, including over 60 members in humans. These GTPases share conserved residues related to nucleotide binding and hydrolysis, and main sequence divergences lie in the carboxyl termini. They cycle between inactive (GDP-bound) and active (GTP-bound) forms and the active site regions, termed Switch I and II, undergo the larger conformational changes between the two states. The Rab11 subfamily members, comprising Rab11a, Rab11b, and Rab25, act in recycling of proteins from the endosomes to the plasma membrane, in transport of molecules from the trans-Golgi network to the plasma membrane and in phagocytosis. In this work, we describe Rab11b-GDP and Rab11b-GppNHp crystal structures solved to 1.55 and 1.95 angstroms resolution, respectively. Although Rab11b shares 90% amino acid identity to Rab11a, its crystal structure shows critical differences relative to previously reported Rab11a structures. Inactive Rab11a formed dimers with unusually ordered Switch regions and missing the magnesium ion at the nucleotide binding site. In this work, inactive Rab11b crystallized as a monomer showing a flexible Switch I and a magnesium ion which is coordinated by four water molecules, the phosphate beta of GDP (beta-P) and the invariant S25. S20 from the P-loop and S42 from the Switch I are associated to GTP hydrolysis rate. In the active structures, S20 interacts with the gamma-P oxygen in Rab11b-GppNHp but does not in Rab11a-GppNHp and the Q70 side chain is found in different positions. In the Rab11a-GTPgammaS structure, S40 is closer to S25 and S42 does not interact with the gamma-P oxygen. These differences indicate that the Rab11 isoforms may possess different GTP hydrolysis rates. In addition, the Switch II of inactive Rab11b presents a 3(10)-helix (residues 69-73) that disappears upon activation. This 3(10)-helix is not found in the Rab11a-GDP structure, which possesses a longer alpha2 helix, spanning from residue 73 to 82 alpha-helix 5.     

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     

Regulation of ENaC expression at the cell surface by Rab11

The epithelial Na⁺ channel (ENaC) is an essential channel responsible for Na⁺ reabsorption. Coexpression of Rab11a and Rab3a small G proteins with ENaC results in a significant increase in channel activity. In contrast, coexpression of Rab5, Rab27a, and Arf-1 had no effect or slightly decreased ENaC activity. Inhibition of MEK with PD98059, Rho-kinase with Y27632 or PI3-kinase with LY294002 had no effect on ENaC activity in Rab11a-transfected CHO cells. Fluorescence imaging methods demonstrate that Rab11a colocalized with ENaC. Rab11a increases ENaC activity in an additive manner with dominant-negative dynamin, which is a GTPase responsible for endocytosis. Brefeldin A, an inhibitor of intracellular protein translocation, blocked the stimulatory action of Rab11a on ENaC activity. We conclude that ENaC channels, present on the apical plasma membrane, are being exchanged with channels from the intracellular pool in a Rab11-dependent manner.     

FIP2 and Rip11 specify Rab11a-mediated cellular distribution of GLUT4 and FAT/CD36 in H9c2-hIR cells

Rab11a has been shown to be involved in different vesicle trafficking processes. To further define the functional role of Rab11a in vesicle movement we knocked down gene expression of Rab11a and two of its effectors, Rip11 and FIP2, in H9c2-hIR cells and measured the cell surface abundance of GLUT4myc and FAT/CD36. We observed that by knocking down Rab11a, both GLUT4myc and FAT/CD36 abundance at the plasma membrane were substantially increased. In the case of GLUT4myc, the in vitro knockdown of FIP2 also increased the cell surface abundance of GLUT4myc. Knockdown of both FIP2 and Rip11 increase the abundance of FAT/CD36 at the plasma membrane. Stimulated translocation of GLUT4myc and FAT/CD36 is not altered after gene knockdown of Rab11a. These data therefore show that (i) Rab11a regulates cell surface abundance of both GLUT4 and FAT/CD36 and that (ii) both Rab11a-dependent processes are differently regulated by Rab11a effector proteins.