Purification, crystallization and X-ray crystallographic analysis of human RAB11(S20V), a constitutively active GTP-binding form.
ABSTRACT: RAB11, a member of the Ras superfamily of small G proteins, is involved in the regulation of vesicle trafficking during endosome recycling. Substitution of Ser20 by Val20 in Rab11 [RAB11(S20V)] inhibits its GTP hydrolysis activity and produces a constitutively active GTP-binding form. In this study, the RAB11(S20V) mutant was overexpressed in Escherichia coli with an engineered C-terminal His tag. RAB11(S20V) was then purified to homogeneity and was crystallized at 293?K. X-ray diffraction data were collected to a resolution of 2.4?Å from a crystal belonging to space group I4, with unit-cell parameters a = 74.11, b = 74.11, c = 149.44?Å. The asymmetric unit was estimated to contain two molecules of RAB11(S20V).
Project description:The small GTPase Rab11 regulates the recycling of endosomes back to the plasma membrane. In its active GTP-bound form, Rab11 binds a novel set of effectors termed the Rab11 family of interacting proteins (Rab11-FIPs) which contain a conserved C-terminal Rab-binding domain (RBD) of unknown structure. Here, a complex of Rab11 with the RBD of Rab11-FIP2 has been purified and crystallized in the trigonal space group P3(1)21, with unit-cell parameters a = 64.99, b = 64.99, c = 112.59 angstroms. Static light-scattering analyses of the molecular weight of the complex in solution are consistent with two copies of Rab11 and two copies of Rab11-FIP2 in the complex.
Project description:We have identified and cloned the cDNA for a 912-aa protein, rab11BP, that interacts with the GTP-containing active form of rab11, a GTP-binding protein that plays a critical role in receptor recycling. Although rab11BP is primarily cytosolic, a significant fraction colocalizes with rab11 in endosomal membranes of both the sorting and recycling subcompartments. In vitro binding of rab11 to native rab11BP requires partial denaturation of the latter to expose an internal binding site located between residues 334 and 504 that is apparently masked by the C-terminal portion of the protein, which includes six repeats known as WD40 domains. Within the cell, rab11BP must undergo a conformational change in which the rab11-binding site becomes exposed, because when coexpressed with rab11 in transfected cells the two proteins formed abundant complexes in association with membranes. Furthermore, although overexpression of rab11BP did not affect transferrin recycling, overexpression of a truncated form of the protein, rab11BP(1-504), that includes the rab11-binding site but lacks the WD40 domains inhibited recycling as strongly as does a dominant negative rab11 mutant protein that does not bind GTP. Strikingly, the inhibition caused by the truncated rab11BP was prevented completely when the cells also expressed a C-terminally deleted, nonprenylatable form of rab11 that, by itself, has no effect on recycling. We propose that rab11BP is an effector for rab11, whose association with this GTP-binding protein is dependent on the action of another membrane-associated factor that promotes the unmasking of the rab11-binding site in rab11BP.
Project description:The recycling endosome localizes to a pericentrosomal region via microtubule-dependent transport. We previously showed that Sec15, an effector of the recycling endosome component, Rab11-GTPase, interacts with the mother centriole appendage protein, centriolin, suggesting an interaction between endosomes and centrosomes. Here we show that the recycling endosome associates with the appendages of the mother (older) centriole. We show that two mother centriole appendage proteins, centriolin and cenexin/ODF2, regulate association of the endosome components Rab11, the Rab11 GTP-activating protein Evi5, and the exocyst at the mother centriole. Development of an in vitro method for reconstituting endosome protein complexes onto isolated membrane-free centrosomes demonstrates that purified GTP-Rab11 but not GDP-Rab11 binds to mother centriole appendages in the absence of membranes. Moreover, centriolin depletion displaces the centrosomal Rab11 GAP, Evi5, and increases mother-centriole-associated Rab11; depletion of Evi5 also increases centrosomal Rab11. This indicates that centriolin localizes Evi5 to centriolar appendages to turn off centrosomal Rab11 activity. Finally, centriolin depletion disrupts recycling endosome organization and function, suggesting a role for mother centriole proteins in the regulation of Rab11 localization and activity at the mother centriole.
Project description:Rab11 is an important protein subfamily in the RabGTPase family. These proteins physiologically function as key regulators of intracellular membrane trafficking processes. Pathologically, Rab11 proteins are implicated in many diseases including cancers, neurodegenerative diseases and type 2 diabetes. Although they are medically important, no previous study has found Rab11 allosteric binding sites where potential drug candidates can bind to. In this study, by employing multiple clustering approaches integrating principal component analysis, independent component analysis and locally linear embedding, we performed structural analyses of Rab11 and identified eight representative structures. Using these representatives to perform binding site mapping and virtual screening, we identified two novel binding sites in Rab11 and small molecules that can preferentially bind to different conformations of these sites with high affinities. After identifying the binding sites and the residue interaction networks in the representatives, we computationally showed that these binding sites may allosterically regulate Rab11, as these sites communicate with switch 2 region that binds to GTP/GDP. These two allosteric binding sites in Rab11 are also similar to two allosteric pockets in Ras that we discovered previously.
Project description:Primary cilia are microtubule-based membrane projections located at the surface of many cells. Defects in primary cilia formation have been implicated in a number of genetic disorders, such as Bardet-Biedl Syndrome and Polycystic Kidney Disease. Recent studies have demonstrated that polarized vesicular transport involving Rab8 and its guanine nucleotide-exchange factor Rabin8 is essential for primary ciliogenesis. Here we report that Rabin8 is a direct downstream effector of Rab11, which functions in membrane trafficking from the trans-Golgi network and recycling endosomes. Rab11, in its GTP-bound form, interacts with Rabin8 and kinetically stimulates the guanine nucleotide-exchange activity of Rabin8 toward Rab8. Rab11 is enriched at the base of the primary cilia and inhibition of Rab11 function by a dominant-negative mutant or RNA interference blocks primary ciliogenesis. Our results suggest that Rab GTPases coordinate with each other in the regulation of vesicular trafficking during primary ciliogenesis.
Project description:Phosphatidylinositol 4-kinasebeta (PI4Kbeta) plays an essential role in maintaining the structural integrity of the Golgi complex. In a search for PI4Kbeta-interacting proteins, we found that PI4Kbeta specifically interacts with the GTP-bound form of the small GTPase rab11. The PI4Kbeta-rab11 interaction is of functional significance because inhibition of rab11 binding to PI4Kbeta abolished the localization of rab11 to the Golgi complex and significantly inhibited transport of vesicular stomatitis virus G protein from the Golgi complex to the plasma membrane. We propose that a novel function of PI4Kbeta is to act as a docking protein for rab11 in the Golgi complex, which is important for biosynthetic membrane transport from the Golgi complex to the plasma membrane.
Project description:Influenza A virus RNA genome exists as eight-segmented ribonucleoprotein complexes containing viral RNA polymerase and nucleoprotein (vRNPs). Packaging of vRNPs and virus budding take place at the apical plasma membrane (APM). However, little is known about the molecular mechanisms of apical transport of newly synthesized vRNP. Transfection of fluorescent-labeled antibody and subsequent live cell imaging revealed that punctate vRNP signals moved along microtubules rapidly but intermittently in both directions, suggestive of vesicle trafficking. Using a series of Rab family protein, we demonstrated that progeny vRNP localized to recycling endosome (RE) in an active/GTP-bound Rab11-dependent manner. The vRNP interacted with Rab11 through viral RNA polymerase. The localization of vRNP to RE and subsequent accumulation to the APM were impaired by overexpression of Rab binding domains (RBD) of Rab11 family interacting proteins (Rab11-FIPs). Similarly, no APM accumulation was observed by overexpression of class II Rab11-FIP mutants lacking RBD. These results suggest that the progeny vRNP makes use of Rab11-dependent RE machinery for APM trafficking.
Project description:The Evi5 oncogene has recently been shown to regulate the stability and accumulation of critical G(1) cell cycle factors including Emi1, an inhibitor of the anaphase-promoting complex/cyclosome, and cyclin A. Sequence analysis of the amino terminus of Evi5 reveals a Tre-2, Bub2, Cdc16 domain, which has been shown to be a binding partner and GTPase-activating protein domain for the Rab family of small Ras-like GTPases. Here we describe the identification of Evi5 as a candidate binding protein for Rab11, a GTPase that regulates intracellular transport and has specific roles in endosome recycling and cytokinesis. By yeast two-hybrid analysis, immunoprecipitation, and Biacore analysis, we demonstrate that Evi5 binds Rab11a and Rab11b in a GTP-dependent manner. However, Evi5 displays no activation of Rab11 GTPase activity in vitro. Evi5 colocalizes with Rab11 in vivo, and overexpression of Rab11 perturbs the localization of Evi5, redistributing it into Rab11-positive recycling endosomes. Interestingly, in vitro binding studies show that Rab11 effector proteins including FIP3 compete with Evi5 for binding to Rab11, suggesting a partitioning between Rab11-Evi5 and Rab11 effector complexes. Indeed, ablation of Evi5 by RNA interference causes a mislocalization of FIP3 at the abscission site during cytokinesis. These data demonstrate that Evi5 is a Rab11 binding protein and that Evi5 may cooperate with Rab11 to coordinate vesicular trafficking, cytokinesis, and cell cycle control independent of GTPase-activating protein function.
Project description:Neuronal pruning is a commonly observed phenomenon for the developing nervous systems to ensure precise wiring of neural circuits. The function of Ik2 kinase and its downstream mediator, Spindle-F (Spn-F), are essential for dendrite pruning of Drosophila sensory neurons during development. However, little is known about how Ik2/Spn-F signaling is transduced in neurons and ultimately results in dendrite pruning. Our genetic analyses and rescue experiments demonstrated that the small GTPase Rab11, especially the active GTP-bound form, is required for dendrite pruning. We also found that Rab11 shows genetic interactions with spn-F and ik2 on pruning. Live imaging of single neurons and antibody staining reveal normal Ik2 kinase activation in Rab11 mutant neurons, suggesting that Rab11 could have a functional connection downstream of and/or parallel to the Ik2 kinase signaling. Moreover, we provide biochemical evidence that both the Ik2 kinase activity and the formation of Ik2/Spn-F/Rab11 complexes are central to promote Rab11 activation in cells. Together, our studies reveal that a critical role of Ik2/Spn-F signaling in neuronal pruning is to promote Rab11 activation, which is crucial for dendrite pruning in neurons.
Project description:Cholesterol, which is endocytosed to the late endosome (LE)/lysosome, is delivered to other organelles through vesicular and nonvesicular transport mechanisms. In this study, we discuss a novel mechanism of cholesterol transport from recycling endosomes (REs) to the trans-Golgi network (TGN) through RELCH/KIAA1468, which is newly identified in this study as a Rab11-GTP- and OSBP-binding protein. After treating cells with 25-hydroxycholesterol to induce OSBP relocation from the cytoplasm to the TGN, REs accumulated around the TGN area, but this accumulation was diminished in RELCH- or OSBP-depleted cells. Cholesterol content in the TGN was decreased in Rab11-, RELCH-, and OSBP-depleted cells and increased in the LE/lysosome. According to in vitro reconstitution experiments, RELCH tethers Rab11-bound RE-like and OSBP-bound TGN-like liposomes and promotes OSBP-dependent cholesterol transfer from RE-like to TGN-like liposomes. These data suggest that RELCH promotes nonvesicular cholesterol transport from REs to the TGN through membrane tethering.