Rab-A2 and Rab-A3 GTPases define a trans-golgi endosomal membrane domain in Arabidopsis that contributes substantially to the cell plate

The Ypt3/Rab11/Rab25 subfamily of Rab GTPases has expanded greatly in Arabidopsis thaliana, comprising 26 members in six provisional subclasses, Rab-A1 to Rab-A6. We show that the Rab-A2 and Rab-A3 subclasses define a novel post-Golgi membrane domain in Arabidopsis root tips. The Rab-A2/A3 compartment was distinct from but often close to Golgi stacks and prevacuolar compartments and partly overlapped the VHA-a1 trans-Golgi compartment. It was also sensitive to brefeldin A and accumulated FM4-64 before prevacuolar compartments did. Mutations in RAB-A2a that were predicted to stabilize the GDP- or GTP-bound state shifted the location of the protein to the Golgi or plasma membrane, respectively. In mitosis, KNOLLE accumulated principally in the Rab-A2/A3 compartment. During cytokinesis, Rab-A2 and Rab-A3 proteins localized precisely to the growing margins of the cell plate, but VHA-a1, GNOM, and prevacuolar markers were excluded. Inducible expression of dominant-inhibitory mutants of RAB-A2a resulted in enlarged, polynucleate, meristematic cells with cell wall stubs. The Rab-A2/A3 compartment, therefore, is a trans-Golgi compartment that communicates with the plasma membrane and early endosomal system and contributes substantially to the cell plate. Despite the unique features of plant cytokinesis, membrane traffic to the division plane exhibits surprising molecular similarity across eukaryotic kingdoms in its reliance on Ypt3/Rab11/Rab-A GTPases.     

Rab Family Proteins Regulate the Endosomal Trafficking and Function of RGS4

RGS4, a heterotrimeric G-protein inhibitor, localizes to plasma membrane (PM) and endosomal compartments. Here, we examined Rab-mediated control of RGS4 internalization and recycling. Wild type and constitutively active Rab5 decreased RGS4 PM levels while increasing its endosomal targeting. Rab5, however, did not appreciably affect the PM localization or function of the M1 muscarinic receptor (M1R)/G_q signaling cascade. RGS4-containing endosomes co-localized with subsets of Rab5-, transferrin receptor-, and Lamp1/Lysotracker-marked compartments suggesting RGS4 traffics through PM recycling or acidified endosome pathways. Rab7 activity promoted TGN association, whereas Rab7(dominant negative) trapped RGS4 in late endosomes. Furthermore, RGS4 was found to co-localize with an endosomal pool marked by Rab11, the protein that mediates recycling/sorting of proteins to the PM. The Cys-12 residue in RGS4 appeared important for its Rab11-mediated trafficking to the PM. Rab11(dominant negative) decreased RGS4 PM levels and increased the number of RGS4-containing endosomes. Inhibition of Rab11 activity decreased RGS4 function as an inhibitor of M1R activity without affecting localization and function of the M1R/G_q signaling complex. Thus, both Rab5 activation and Rab11 inhibition decreased RGS4 function in a manner that is independent from their effects on the localization and function of the M1R/G_q signaling complex. This is the first study to implicate Rab GTPases in the intracellular trafficking of an RGS protein. Thus, Rab GTPases may be novel molecular targets for the selective regulation of M1R-mediated signaling via their specific effects on RGS4 trafficking and function.     

Rab4 affects both recycling and degradative endosomal trafficking

The small GTPases Rab4, Rab5 and Rab7 are endosomal proteins which play important roles in the regulation of various stages of endosomal trafficking. Rab4 and Rab5 have both been localized to early endosomes and have been shown to control recycling and endosomal fusion, respectively. Rab7, a marker of the late endosomal compartment, is involved in the regulation of the late endocytic pathway. Here, we compare the role of Rab4, Rab5 and Rab7 in early and late endosomal trafficking in HeLa cells monitoring ligand uptake, recycling and degradation. Expression of the Rab4 dominant negative mutant (Rab4AS22N) leads to a significant reduction in both recycling and degradation while, as expected, Rab7 mutants exclusively affect epidermal growth factor (EGF) and low density lipoprotein degradation. As also expected, expression of the dominant negative Rab5 mutant perturbs internalization kinetics and affects both recycling and degradation. Expression of Rab4WT and dominant positive mutant (Rab4AQ67L) changes dramatically the morphology of the transferrin compartment leading to the formation of membrane tubules. These transferrin positive tubules display swellings (varicosities) some of which are positive for early endosomal antigen-1 and contain EGF. We propose that the Rab4GTPase is important for the function of the early sorting endosomal compartment, affecting trafficking along both recycling and degradative pathways.     

beta 2-adrenergic receptor internalization, endosomal sorting, and plasma membrane recycling are regulated by rab GTPases

Rab GTPases are recognized as critical regulatory factors involved in vesicular membrane transport and endosomal fusion. For example, Rab5 directs the transport and fusion of endocytic vesicles to and with early endosomes, whereas Rab4 is thought to control protein trafficking from early endosomes back to the plasma membrane. In the present study, we investigated the role of Rab5 and Rab4 GTPases in regulating the endocytosis, intracellular sorting, and the plasma membrane recycling of the beta(2)AR. In cells expressing the dominant-negative Rab5-S34N mutant, beta(2)AR internalization was impaired, and beta(2)AR-bearing endocytic vesicles remained in either close juxtaposition or physically attached to the plasma membrane. In contrast, a constitutively active Rab5-Q79L mutant redirected internalized beta(2)AR to enlarged endosomes but did not prevent beta(2)AR dephosphorylation and recycling. The expression of either wild-type Rab4 or a Rab4-N121I mutant did not prevent beta(2)AR dephosphorylation. However, the dominant-negative Rab4-N121I mutant blocked beta(2)AR resensitization by blocking receptor recycling from endosomes back to the cell surface. Our data indicate that, in addition to regulating the intracellular trafficking and fusion of beta(2)AR-bearing endocytic vesicles, Rab5 also contributes to the formation and/or budding of clathrin-coated vesicles. Furthermore, beta(2)AR dephosphorylation occurs as the receptor transits between Rab5- and Rab4-positive compartments.     

Regulation of epithelial ion channels by Rab GTPases

Epithelial ion channels are crucial to many of life's processes and disruption of their functions can lead to several disorders. Cystic fibrosis, an autosomal recessive disorder, is caused by defects in the biosynthesis or function of the CFTR chloride channel. Similarly, mutations in certain ENaC genes leading to increased or reduced channel activity cause diseases such as Liddle's syndrome or PHA. In order for ion channel proteins to be functional they need to be expressed on the plasma membrane. Thus, molecules that modulate the trafficking of ion channels to and from the membrane are of utmost significance. Among the numerous factors that regulate their functioning is a family of small GTPases known as Rab proteins. While Rabs have always played a pivotal role in membrane trafficking, their diversity of functions and plethora of interacting partners have lately been brought to light. Recent studies reveal that multiple Rab isoforms physically interact with and/or modulate the activity of several ion channels. Rab proteins have the ability to serve as molecular switches and many of the ion channels are regulated differentially by the GTP- or GDP-bound Rab isoforms. This review examines the role of Rab GTPases in the trafficking of ion channels, including CFTR, ENaC, TRPV5/6, and aquaporins, based on recent evidence.     

Rab GTPases Regulate Endothelial Cell Protein C Receptor-Mediated Endocytosis and Trafficking of Factor VIIa

Recent studies have established that factor VIIa (FVIIa) binds to the endothelial cell protein C receptor (EPCR). FVIIa binding to EPCR may promote the endocytosis of this receptor/ligand complex. Rab GTPases are known to play a crucial role in the endocytic and exocytic pathways of receptors or receptor/ligand complexes. The present study was undertaken to investigate the role of Rab GTPases in the intracellular trafficking of EPCR and FVIIa. CHO-EPCR cells and human umbilical vein endothelial cells (HUVEC) were transduced with recombinant adenoviral vectors to express wild-type, constitutively active, or dominant negative mutant of various Rab GTPases. Cells were exposed to FVIIa conjugated with AF488 fluorescent probe (AF488-FVIIa), and intracellular trafficking of FVIIa, EPCR, and Rab proteins was evaluated by immunofluorescence confocal microscopy. In cells expressing wild-type or constitutively active Rab4A, internalized AF488-FVIIa accumulated in early/sorting endosomes and its entry into the recycling endosomal compartment (REC) was inhibited. Expression of constitutively active Rab5A induced large endosomal structures beneath the plasma membrane where EPCR and FVIIa accumulated. Dominant negative Rab5A inhibited the endocytosis of EPCR-FVIIa. Expression of constitutively active Rab11 resulted in retention of accumulated AF488-FVIIa in the REC, whereas expression of a dominant negative form of Rab11 led to accumulation of internalized FVIIa in the cytoplasm and prevented entry of internalized FVIIa into the REC. Expression of dominant negative Rab11 also inhibited the transport of FVIIa across the endothelium. Overall our data show that Rab GTPases regulate the internalization and intracellular trafficking of EPCR-FVIIa.     

Molecular characterization of Rab11 interactions with members of the family of Rab11-interacting proteins

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 GTP-bound 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 GDP-bound Rab11 interactions to occur in vivo. Mutational analysis revealed that both the conserved hydrophobic patch and Tyr628 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.     

Plant Rabs: Characterization,Functional Diversity,and Role in Stress Tolerance

Rab proteins form the largest family of small guanosine triphosphate (GTP)-binding proteins. The Rab family in plants is divided into eight subfamilies, Rab1, Rab2, Rab5, Rab6, Rab7, Rab8, Rab11, and Rab18. Phylogenetic analyses of amino acid sequence of Rab GTPases suggest their segregation into subfamilies on the basis of their localization and/or function in membrane trafficking. The Rab GTPases are localized to the cytosolic face of specific intracellular membranes, where they function as regulators of distinct steps in membrane-trafficking pathways. The Rab proteins show highly conserved structural features with a great functional versatility. They play an important role in regulating hormone signaling during fruit ripening and apical dominance, brassinosteroid biosynthesis, pollen and nodule development, and in response to both abiotic and biotic stresses.     

Pairing phosphoinositides with calcium ions in endolysosomal dynamics: phosphoinositides control the direction and specificity of membrane trafficking by regulating the activity of calcium channels in the endolysosomes

The direction and specificity of endolysosomal membrane trafficking is tightly regulated by various cytosolic and membrane-bound factors, including soluble NSF attachment protein receptors (SNAREs), Rab GTPases, and phosphoinositides. Another trafficking regulatory factor is juxta-organellar Ca(2+) , which is hypothesized to be released from the lumen of endolysosomes and to be present at higher concentrations near fusion/fission sites. The recent identification and characterization of several Ca(2+) channel proteins from endolysosomal membranes has provided a unique opportunity to examine the roles of Ca(2+) and Ca(2+) channels in the membrane trafficking of endolysosomes. SNAREs, Rab GTPases, and phosphoinositides have been reported to regulate plasma membrane ion channels, thereby suggesting that these trafficking regulators may also modulate endolysosomal dynamics by controlling Ca(2+) flux across endolysosomal membranes. In this paper, we discuss the roles of phosphoinositides, Ca(2+) , and potential interactions between endolysosomal Ca(2+) channels and phosphoinositides in endolysosomal dynamics.     

From sorting endosomes to exocytosis: association of Rab4 and Rab11 GTPases with the Fc receptor, FcRn, during recycling

A longstanding question in cell biology is how is the routing of intracellular organelles within cells regulated? Although data support the involvement of Rab4 and Rab11 GTPases in the recycling pathway, the function of Rab11 in particular is uncertain. Here we have analyzed the association of these two Rab GTPases with the Fc receptor, FcRn, during intracellular trafficking. This Fc receptor is both functionally and structurally distinct from the classical Fcgamma receptors and transports immunoglobulin G (IgG) within cells. FcRn is therefore a recycling receptor that sorts bound IgG from unbound IgG in sorting endosomes. In the current study we have used dual color total internal reflection fluorescence microscopy (TIRFM) and wide-field imaging of live cells to analyze the events in human endothelial cells that are involved in the trafficking of FcRn positive (FcRn(+)) recycling compartments from sorting endosomes to exocytic sites at the plasma membrane. Our data are consistent with the following model for this pathway: FcRn leaves sorting endosomes in Rab4(+)Rab11(+) or Rab11(+) compartments. For Rab4(+)Rab11(+) compartments, Rab4 depletion occurs by segregation of the two Rab proteins into discrete domains that can separate. The Rab11(+)FcRn(+) vesicle or tubule subsequently fuses with the plasma membrane in an exocytic event. In contrast to Rab11, Rab4 is not involved in exocytosis.     

Endosomal trafficking of the G protein-coupled receptor somatostatin receptor 3

Intracellular trafficking of G protein-coupled receptors (GPCRs) regulates their surface availability and determines cellular response to agonists. Rab GTPases regulate membrane trafficking and identifying Rab networks controlling GPCR trafficking is essential for understanding GPCR signaling. We used real time imaging to show that somatostatin receptor 3 (SSTR3) traffics through Rab4-, Rab21-, and Rab11-containing endosomes, but largely bypasses Rab5 and Rab7 endosomes. We show that SSTR3 rapidly traffics through Rab4 endosomes but moves slower through Rab21 and Rab11 endosomes. SSTR3 passage through each endosomal compartment is regulated by the cognate Rab since expression of the inactive Rab4/S22N, Rab21/T33N, and Rab11/S25N inhibits SSTR3 trafficking. Thus, Rab4, Rab21, and Rab11 may represent therapeutic targets to modulate surface availability of SSTR3 for agonist binding. Our novel finding that Rab21 regulates SSTR3 trafficking suggests that Rab21 may play a role in trafficking of other GPCRs.     

Small GTPase Rab17 regulates dendritic morphogenesis and postsynaptic development of hippocampal neurons

Neurons are compartmentalized into two morphologically, molecularly, and functionally distinct domains: axons and dendrites, and precise targeting and localization of proteins within these domains are critical for proper neuronal functions. It has been reported that several members of the Rab family small GTPases that are key mediators of membrane trafficking, regulate axon-specific trafficking events, but little has been elucidated regarding the molecular mechanisms that underlie dendrite-specific membrane trafficking. Here we show that Rab17 regulates dendritic morphogenesis and postsynaptic development in mouse hippocampal neurons. Rab17 is localized at dendritic growth cones, shafts, filopodia, and mature spines, but it is mostly absent in axons. We also found that Rab17 mediates dendrite growth and branching and that it does not regulate axon growth or branching. Moreover, shRNA-mediated knockdown of Rab17 expression resulted in a dramatically reduced number of dendritic spines, probably because of impaired filopodia formation. These findings have revealed the first molecular link between membrane trafficking and dendritogenesis.     

Arf1 orchestrates Rab GTPase conversion at the trans-Golgi network

Rab family GTPases are key organizers of membrane trafficking and function as markers of organelle identity. Accordingly, Rab GTPases often occupy specific membrane domains, and mechanisms exist to prevent the inappropriate mixing of distinct Rab domains. The yeast Golgi complex can be divided into two broad Rab domains: Ypt1 (Rab1) and Ypt6 (Rab6) are present at the early/medial Golgi and sharply transition to Ypt31/32 (Rab11) at the late Golgi/trans-Golgi network (TGN). This Rab conversion has been attributed to GTPase-activating protein (GAP) cascades in which Ypt31/32 recruits the Rab-GAPs Gyp1 and Gyp6 to inactivate Ypt1 and Ypt6, respectively. Here we report that Rab transition at the TGN involves additional layers of regulation. We provide new evidence confirming the TRAPPII complex as an important regulator of Ypt6 inactivation and uncover an unexpected role of the Arf1 GTPase in recruiting Gyp1 to drive Ypt1 inactivation at the TGN. Given its established role in directly recruiting TRAPPII to the TGN, Arf1 is therefore a master regulator of Rab conversion on maturing Golgi compartments.     

Arabidopsis TRAPPII is functionally linked to Rab-A,but not Rab-D in polar protein trafficking in trans-Golgi network

The trans-Golgi network (TGN) in plant cells is an independent organelle, displaying rapid association and dissociation with Golgi bodies. In plant cells, the TGN is the site where secretory and endocytic membrane trafficking meet. Cell wall components, signaling molecules and auxin transporters have been found to undergo intracellular trafficking around the TGN. However, how different trafficking pathways are regulated and how different cargoes are sorted in the TGN is poorly defined in plant cells. Using a combined approach of genetic and in vivo imaging, we recently demonstrated that Arabidopsis TRAPPII acts in the TGN and is required for polar targeting of PIN2, but not PIN1, auxin efflux carrier in root tip cells. Here, we report that, TRAPPII in Arabidopsis is required for polar distribution of AUX1, an auxin influx carrier in protophloem cells and epidermal cells of Arabidopsis root tips. In yeast cells, TRAPPII serves as a guanine-nucleotide exchange factor (GEF) for Ypt1 and Ypt31/32 in late Golgi trafficking, while in mammalian cells, TRAPPII acts as a GEF for Rab1 (homolog of yeast Ypt1) in early Golgi trafficking. We show here that TRAPPII in Arabidopsis is functionally linked to Rab-A proteins, homologs of yeast Ypt31/32, but not Rab-D proteins, homologs of yeast Ypt1 and animal Rab1 proteins.     

D-AKAP2 Interacts with Rab4 and Rab11 through Its RGS Domains and Regulates Transferrin Receptor Recycling

Dual-specific A-kinase-anchoring protein 2 (D-AKAP2/AKAP10), which interacts at its carboxyl terminus with protein kinase A and PDZ domain proteins, contains two tandem regulator of G-protein signaling (RGS) domains for which the binding partners have remained unknown. We show here that these RGS domains interact with Rab11 and GTP-bound Rab4, the first demonstration of RGS domains binding small GTPases. Rab4 and Rab11 help regulate membrane trafficking through the endocytic recycling pathways by recruiting effector proteins to specific membrane domains. Although D-AKAP2 is primarily cytosolic in HeLa cells, a fraction of the protein localizes to endosomes and can be recruited there to a greater extent by overexpression of Rab4 or Rab11. D-AKAP2 also regulates the morphology of the Rab11-containing compartment, with co-expression causing accumulation of both proteins on enlarged endosomes. Knockdown of D-AKAP2 by RNA interference caused a redistribution of both Rab11 and the constitutively recycling transferrin receptor to the periphery of cells. Knockdown also caused an increase in the rate of transferrin recycling, suggesting that D-AKAP2 promotes accumulation of recycling proteins in the Rab4/Rab11-positive endocytic recycling compartment.     

The GTP-Bound form of Rab3D Promotes Lipid Droplet Growth in Adipocyte

Molecular Biology - Rab GTPases are major regulators of membrane trafficking. Proteome profiling reveals dozens of Rab proteins associated with lipid droplets (LDs), but few of them have been...     

The novel Rab11-FIP/Rip/RCP family of proteins displays extensive homo- and hetero-interacting abilities

The Rab11-FIP/Rip/RCP proteins are a recently described novel protein family, whose members interact with Rab GTPases that function in endosomal recycling. To date, five such proteins have been described in humans, all of which interact with Rab11, and one (RCP) also interacts with Rab4. Here, we characterise several of these proteins with respect to their ability to interact with Rab4, as well as their ability to self-interact, and to interact with each other. We now demonstrate that two of the family members-pp75/Rip11 and Rab11-FIP3 do not bind Rab4 and show that several members of the family can self-interact and interact with each other. These interactions primarily involve their C-terminal end which includes the Rab binding domain (RBD) that is contained within a predicted coiled-coil, or ERM motif. We identify a new (sixth) member of the protein family, which we propose to name Rab11-FIP4, and report the family evolutionary complexity and chromosomal distribution. Furthermore, we propose that the ability of these proteins to bind each other will be important in effecting membrane trafficking events by forming protein 'platforms,' regulated by Rab11 and/or Rab4 activity.     

Rab11 GTPase-regulated membrane trafficking is crucial for tip-focused pollen tube growth in tobacco

Pollen tube growth is a polarized growth process whereby the tip-growing tubes elongate within the female reproductive tissues to deliver sperm cells to the ovules for fertilization. Efficient and regulated membrane trafficking activity incorporates membrane and deposits cell wall molecules at the tube apex and is believed to underlie rapid and focused growth at the pollen tube tip. Rab GTPases, key regulators of membrane trafficking, are candidates for important roles in regulating pollen tube growth. We show that a green fluorescent protein-tagged Nicotiana tabacum pollen-expressed Rab11b is localized predominantly to an inverted cone-shaped region in the pollen tube tip that is almost exclusively occupied by transport vesicles. Altering Rab11 activity by expressing either a constitutive active or a dominant negative variant of Rab11b in pollen resulted in reduced tube growth rate, meandering pollen tubes, and reduced male fertility. These mutant GTPases also inhibited targeting of exocytic and recycled vesicles to the pollen tube inverted cone region and compromised the delivery of secretory and cell wall proteins to the extracellular matrix. Properly regulated Rab11 GTPase activity is therefore essential for tip-focused membrane trafficking and growth at the pollen tube apex and is pivotal to reproductive success.     

Recycling and resensitization of the neurokinin 1 receptor. Influence of agonist concentration and Rab GTPases

Substance P (SP) induces endocytosis and recycling of the neurokinin 1 receptor (NK1R) in endothelial cells and spinal neurons at sites of inflammation and pain, and it is thus important to understand the mechanism and function of receptor trafficking. We investigated how the SP concentration affects NK1R trafficking and determined the role of Rab GTPases in trafficking. NK1R trafficking was markedly influenced by the SP concentration. High SP (10 nM) induced translocation of the NK1R and beta-arrestin 1 to perinuclear sorting endosomes containing Rab5a, where NK1R remained for >60 min. Low SP (1 nM) induced translocation of the NK1R to early endosomes located immediately beneath the plasma membrane that also contained Rab5a and beta-arrestin 1, followed by rapid recycling of the NK1R. Overexpression of Rab5a promoted NK1R translocation to perinuclear sorting endosomes, whereas the GTP binding-deficient mutant Rab5aS34N caused retention of the NK1R in superficial early endosomes. NK1R translocated from superficial early endosomes to recycling endosomes containing Rab4a and Rab11a, and Rab11aS25N inhibited NK1R recycling. Rapid NK1R recycling coincided with resensitization of SP-induced Ca2+ mobilization and with the return of surface SP binding sites. Resensitization was minimally affected by inhibition of vacuolar H(+)-ATPase and phosphatases but was markedly suppressed by disruption of Rab4a and Rab11a. Thus, whereas beta-arrestins mediate NK1R endocytosis, Rab5a regulates translocation between early and sorting endosomes, and Rab4a and Rab11a regulate trafficking through recycling endosomes. We have thus identified a new function of Rab5a as a control protein for directing concentration-dependent trafficking of the NK1R into different intracellular compartments and obtained evidence that Rab4a and Rab11a contribute to G-protein-coupled receptor recycling from early endosomes.     

Small GTPases in vesicle trafficking

Plant small GTPases belonging to the Rop, Arf, and Rab families are regulators of vesicle trafficking. Rop GTPases regulate actin dynamics and modulate H(2)O(2) production in polar cell growth and pathogen defence. A candidate Rop GDP to Rop GTP exchange factor (RopGEF) SPIKE1 is involved in the morphogenesis of leaf epidermal cells. The ArfGEF GNOM regulates the endosomal recycling of the PIN proteins, which are involved in polar auxin transport. Intracellular localisation of small GTPases and functional studies using dominant mutant versions of Arf and Rab GTPases are defining novel plant-specific membrane compartments, especially those that participate in endosomal vesicle trafficking.