Structure of the extremely slow GTPase Rab6A in the GTP bound form at 1.8A resolution

Rab/Ypt GTPases represent a>60 member large family of membrane traffic regulators in eukaryotic cells. Members of this group display intrinsic GTPase activity varying over two orders of magnitude. Here, we show that Rab6A represents the RabGTPase with the slowest spontaneous GTPase activity yet measured (5x10(-6)s(-1)). Due to the very low intrinsic hydrolysis rate we were able to crystallise and solve the structure of the Rab6A:GTP complex to 1.82A resolution. Analysis of the structure suggests that low catalytic activity of the Rab6A might be due to high flexibility of the Switch II region and a low degree of constraint of critically important for catalysis Gln 72.     

Rab6 family proteins interact with the dynein light chain protein DYNLRB1

The small GTPase Rab6 is a key regulator in the retrograde transfer from endosomes via the Golgi to the ER. Three isoforms of Rab6 have been identified, the ubiquitously expressed Rab6A and Rab6A', and the brain specific Rab6B. Recent studies have shown that Rab6A' is the major isoform regulating this retrograde transport. Cytoplasmic dynein is the main motor protein complex for this transport. Dynein consists of two heavy chains, two intermediate chains, four light intermediate chains and several light chains, called roadblock/LC7 proteins or DYNLRB proteins. In mammalian cells two light chain isoforms have been identified, DYNLRB1 and DYNLRB2. We here show with yeast-two-hybrid, co-immunoprecipitation and pull down studies that DYNLRB1 specifically interacts with all three Rab6 isoforms and co-localises at the Golgi. This is the first example of a direct interaction between Rab6 isoforms and the dynein complex. Pull down experiments showed further preferred association of DYNLRB1 with GTP-bound Rab6A and interestingly GDP-bound Rab6A' and Rab6B. In addition DYNLRB1 was found in the Golgi apparatus where it co-localises with EYFP-Rab6 isoforms. DYNLRB is a putative modulator of the intrinsic GTPase activity of GTP-binding proteins. In vitro we were not able to reproduce this effect on Rab6 GTPase activity.     

Tyrosine phosphorylation of the Rab24 GTPase in cultured mammalian cells

Several members of the large family of Rab GTPases have been shown to function in vesicular trafficking in mammalian cells. However, the exact role of Rab24 remains poorly defined. Rab24 differs from other Rab proteins in that it has a low intrinsic GTPase activity and is not efficiently prenylated. Here we report an additional unique property of Rab24; i.e., the protein can undergo tyrosine phosphorylation when overexpressed in cultured cells. Immunoblot analyses with specific anti-phosphotyrosine monoclonal antibodies revealed the presence of phosphotyrosine (pTyr) on myc-Rab24 in whole cell lysates and immunoprecipitated samples. No pTyr was detected on other overexpressed myc-tagged GTPases (H-Ras, Rab1b, Rab6, Rab11 or Rab13). Comparisons of myc-Rab24 in the soluble and particulate fractions from HEK293 and HEp-2 cells indicated that the cytosolic pool of Rab24 was more heavily phosphorylated than the membrane pool. Treatment of transfected cells with the broad-spectrum tyrosine kinase inhibitor, genistein, as well as the specific Src-family kinase inhibitor, PP2, eliminated the pTyr signal from Rab24. In contrast the receptor tyrosine kinase inhibitor, tyrphostin A25, had no effect. Tyrosine phosphorylation of Rab24 was reduced by alanine substitution of two unique tyrosines, one found in a strong consensus phosphorylation motif (Y [Formula: see text] ) in the hypervariable domain (Y172) and the other falling within the GXXXGK(S/T) motif known as the P-loop (Y17). The latter region is known to influence GTP hydrolysis in Rab proteins, so the phosphorylation of Y17 could contribute to the low intrinsic GTPase activity of Rab24. This is the first report of tyrosine phosphorylation in any member of the Ras superfamily and it raises the possibility that this type of modification could influence Rab24 targeting and interactions with effector protein complexes.     

Amino acid residues in the Ras-like GTPase Rab3A that specify sensitivity to factors that regulate the GTP/GDP cycling of Rab3A.

Two cellular factors have been described, Rab3A-GAP (GTPase-activating protein) and Rab3A-GRF (guanine nucleotide releasing factor) which, respectively, accelerate the intrinsic GTPase activity of, or the rate of dissociation of GDP from, the Ras-related GTP-binding protein, p25 Rab3A. Mutational analysis of p25 Rab3A was undertaken to define amino acid residues important for interaction with these factors. Mutations in residues 51-59, which correspond to the effector domain of p21 Ras, completely abolished sensitivity of p25 Rab3A to Rab3A-GRF and decreased the affinity of p25 Rab3A for Rab3A-GRF. Surprisingly, only one mutant in this region was Rab3A-GAP-insensitive, while the others retained partial, complete, or significantly increased GAP responsiveness. Mutations in the first G-domain had only modest effects on intrinsic GTPase activity and little effect on either Rab3A-GRF or Rab3A-GAP interactions. Truncation of 34 residues from the carboxyl terminus had no effect Rab3A-GAP sensitivity but facilitated Rab3A-GRF stimulation. Mutation T36N, analogous to the dominant inhibitory mutation T17N in Ras, which has been hypothesized to sequester an upstream activator of Ras, conferred a 10-fold higher affinity upon p25 Rab3A for Rab3A-GRF.     

Mutant Rab24 GTPase is targeted to nuclear inclusions

Background  

Members of the Rab GTPase family regulate intracellular protein trafficking, but the specific function of Rab24 remains unknown. Several attributes distinguish this protein from other members of the Rab family, including a low intrinsic GTPase activity.     

Biochemical characterization of Rab3-GTPase-activating protein reveals a mechanism similar to that of Ras-GAP

Small G proteins of the Rab family are regulators of intracellular vesicle traffic. Their intrinsic rate of GTP hydrolysis is very low but is enhanced by specific GTPase-activating proteins (GAPs) that switch G proteins to their inactive form. We have characterized the activity of recombinant Rab3-GAP on Rab3A in solution. The K(m) and K(d) values (75 microm) indicate a low affinity of Rab3-GAP for its substrate. The affinity is higher for the transition state analog Rab3A:GDP:AlF(x) (15 microm). The k(cat) (1 s(-)(1)) is within the range of values reported for other GAPs. A mutation in the switch I region of Rab3A disrupted the interaction with Rab3-GAP. Furthermore, Rabphilin, a putative target of Rab3, inhibited the activity of Rab3-GAP on Rab3. Therefore, the Rab3-GAP-binding site involves the switch I region of Rab3 and overlaps with the Rabphilin-binding domain. Substitution of a single arginine residue (Arg-728) of Rab3-GAP disrupted its catalytic activity but not its interaction with Rab3A. We propose that Rab3-GAP, like Ras- and Rho-GAPs, stabilizes the transition state of Rab3 and provides a critical arginine residue to accelerate the GTPase reaction.     

Crystal structure of the intraflagellar transport complex 25/27

The cilium is an important organelle that is found on many eukaryotic cells, where it serves essential functions in motility, sensory reception and signalling. Intraflagellar transport (IFT) is a vital process for the formation and maintenance of cilia. We have determined the crystal structure of Chlamydomonas reinhardtii IFT25/27, an IFT sub‐complex, at 2.6 Å resolution. IFT25 and IFT27 interact via a conserved interface that we verify biochemically using structure‐guided mutagenesis. IFT27 displays the fold of Rab‐like small guanosine triphosphate hydrolases (GTPases), binds GTP and GDP with micromolar affinity and has very low intrinsic GTPase activity, suggesting that it likely requires a GTPase‐activating protein (GAP) for robust GTP turnover. A patch of conserved surface residues contributed by both IFT25 and IFT27 is found adjacent to the GTP‐binding site and could mediate the binding to other IFT proteins as well as to a potential GAP. These results provide the first step towards a high‐resolution structural understanding of the IFT complex.     

TBC domain family, member 15 is a novel mammalian Rab GTPase-activating protein with substrate preference for Rab7

Ypt/Rabs are Ras-related GTPases that function as key regulators of intracellular vesicular trafficking. Their slow intrinsic rates of GTP hydrolysis are catalyzed by GTPase-activating proteins (GAPs). Ypt/Rab-GAPs constitute a family of proteins that contain a TBC (Tre-2/Bub2/Cdc16) domain. Only three of the 51 family members predicted in the human genome are confirmed Ypt/Rab-GAPs. Here, we report the identification and characterization of a novel mammalian Ypt/Rab-GAP, TBC domain family, member 15 (TBC1D15). TBC1D15 is ubiquitously expressed and localized predominantly to the cytosol. The TBC domain of TBC1D15 exhibits relatively high homology with that of Gyp7p, a yeast Ypt/Rab-GAP. Furthermore, TBC1D15 stimulates the intrinsic GTPase activity of Rab7, and to a lesser extent Rab11, but is essentially inactive towards Rab4 or Rab6. These data increase the number of mammalian TBC domain family members with demonstrated Rab-GAP activity to four, and suggest that TBC1D15 may be involved in Rab7-mediated late endosomal trafficking.     

Molecular mechanism of Ras-related protein Rab-5A and effect of mutations in the catalytically active phosphate-binding loop

Ras-related protein (Rab-5a) is primarily involved in the regulation of early endosome fusion during endocytosis and takes part in the budding process. During GTP hydrolysis, Rab5a was spotted in the cytoplasmic side of early endosomes in association with the GTP. Previous study suggested that the substitution of alanine with proline at position 30 of Rab5a reduces the GTPase activity around 12-fold, while, with arginine substitution stimulates the intrinsic GTP hydrolysis by 5-fold. Most of the other substitutions at this position show a little or no effect on the GTPase activity. In this paper, structure analysis and molecular dynamics (MD) simulation studies of human Rab5a and its mutants have been extensively carried out. The effect of binding of a non-hydrolyzable GTP analog guanosine-5′-(β, γ)-imidotriphosphate (GppNHp) with Rab5a and its mutants are described. The objective of the current study is to perform a detailed examination of structural flexibility of Rab5a and its mutants p.Ala30Pro and p.Ala30Arg using MD simulations. Our observations suggest that mutant p.Ala30Arg stabilize the protein molecule when bound to GppNHp which offers additional contacts. Despite an in silico approach, this study provides a deep insight into the impact of mutation on the structure, function, stability, and mechanism of binding of GppNHp to the Rab5a at molecular level.     

Structural basis of activation and GTP hydrolysis in Rab proteins

BACKGROUND: Rab proteins comprise a large family of GTPases that regulate vesicle trafficking. Despite conservation of critical residues involved in nucleotide binding and hydrolysis, Rab proteins exhibit low sequence identity with other GTPases, and the structural basis for Rab function remains poorly characterized. RESULTS: The 2. 0 A crystal structure of GppNHp-bound Rab3A reveals the structural determinants that stabilize the active conformation and regulate GTPase activity. The active conformation is stabilized by extensive hydrophobic contacts between the switch I and switch II regions. Serine residues in the phosphate-binding loop (P loop) and switch I region mediate unexpected interactions with the gamma phosphate of GTP that have not been observed in previous GTPase structures. Residues implicated in the interaction with effectors and regulatory factors map to a common face of the protein. The electrostatic potential at the surface of Rab3A indicates a non-uniform distribution of charged and nonpolar residues. CONCLUSIONS: The major structural determinants of the active conformation involve residues that are conserved throughout the Rab family, indicating a common mode of activation. Novel interactions with the gamma phosphate impose stereochemical constraints on the mechanism of GTP hydrolysis and provide a structural explanation for the large variation of GTPase activity within the Rab family. An asymmetric distribution of charged and nonpolar residues suggests a plausible orientation with respect to vesicle membranes, positioning predominantly hydrophobic surfaces for interaction with membrane-associated effectors and regulatory factors. Thus, the structure of Rab3A establishes a framework for understanding the molecular mechanisms underlying the function of Rab GTPases.     

Functional characterization of two RAB27A missense mutations found in Griscelli syndrome type 2

Human Griscelli syndrome type 2 (GS-2) is characterized by partial albinism and a severe immunologic disorder as a result of RAB27A mutations. In melanocytes, Rab27A forms a tripartite complex with a specific effector Slac2-a/melanophilin and myosin Va, and the complex regulates melanosome transport. Here, we report a novel homozygous missense mutation of Rab27A, i.e. K22R, in a Persian GS-2 patient and the results of analysis of the impact of the K22R mutation and the previously reported I44T mutation on protein function. Both mutations completely abolish Slac2-a/melanophilin binding activity but they affect the biochemical properties of Rab27A differently. The Rab27A(K22R) mutant lacks the GTP binding ability and exhibits cytosolic localization in melanocytes. By contrast, neither intrinsic GTPase activity nor melanosomal localization of Rab27A is affected by the I44T mutation, but the Rab27A(I44T) mutant is unable to recruit Slac2-a/melanophilin. Interestingly, the two mutations differently affect binding to other Rab27A effectors, Slp2-a, Slp4-a/granuphilin-a, and Munc13-4. The Rab27A(K22R) mutant normally binds Munc13-4, but not Slp2-a or Slp4-a, whereas the Rab27A(I44T) mutant shows reduced binding activity to Slp2-a and Munc13-4 but normally binds Slp4-a.     

Comprehensive Screening for Novel Rab‐Binding Proteins by GST Pull‐Down Assay Using 60 Different Mammalian Rabs‡

The Rab family belongs to the Ras‐like small GTPase superfamily and is implicated in membrane trafficking through interaction with specific effector molecules. Because of the large number of Rab isoforms in mammals, however, the effectors of most of the mammalian Rabs are yet to be identified. In this study, we systematically screened five different cell or tissue lysates for novel Rab effectors by a combination of glutathione S‐transferase (GST) pull‐down assay with 60 different mammalian Rabs and mass spectroscopic analysis. Three of the 21 Rab‐binding proteins we identified, mKIAA1055/TBC1D2B (Rab22‐binding protein), GAPCenA/TBC1D11 (Rab36‐binding protein) and centaurin β2/ACAP2 (Rab35‐binding protein), are GTPase‐activating proteins (GAPs) for Rab or Arf. Although it has recently been proposed that the Rab–GAP (Tre‐2 /Bub2/Cdc16) domain physically interacts with its substrate Rab, these three GAPs interacted with specific Rabs via a domain other than a GAP domain, e.g. centaurin β2 binds GTP‐Rab35 via the ankyrin repeat (ANKR) domain. Although centaurin β2 did not exhibit any Rab35–GAP activity in vitro, the Rab35‐binding ANKR domain of centaurin β2 was found to be required for its plasma membrane localization and regulation of Rab35‐dependent neurite outgrowth of PC12 cells through inactivation of Arf6. These findings suggest a novel mode of interaction between Rab and GAP.     

Structural plasticity of an invariant hydrophobic triad in the switch regions of Rab GTPases is a determinant of effector recognition

Rab GTPases function as regulatory components of an evolutionarily conserved machinery that mediates docking, priming, and fusion of vesicles with intracellular membranes. We have previously shown that the active conformation of Rab3A is stabilized by a substantial hydrophobic interface between the putative conformational switch regions (Dumas, J. J., Zhu, Z., Connolly, J. L., and Lambright, D. G. (1999) Structure 7, 413-423). A triad of invariant hydrophobic residues at this switch interface (Phe-59, Trp-76, and Tyr-91) represents a major interaction determinant between the switch regions of Rab3A and the Rab3A-specific effector Rabphilin3A (Ostermeier, C., and Brunger, A. T. (1999) Cell 96, 363-374). Here, we report the crystal structure of the active form of Rab5C, a prototypical endocytic Rab GTPase. As is true for Rab3A, the active conformation of Rab5C is stabilized by a hydrophobic interface between the switch regions. However, the conformation of the invariant hydrophobic triad (residues Phe-58, Trp-75, and Tyr-90 in Rab5C) is dramatically altered such that the resulting surface is noncomplementary to the switch interaction epitope of Rabphilin3A. This structural rearrangement reflects a set of nonconservative substitutions in the hydrophobic core between the central beta sheet and the alpha2 helix. These observations demonstrate that structural plasticity involving an invariant hydrophobic triad at the switch interface contributes to the mechanism by which effectors recognize distinct Rab subfamilies. Thus, the active conformation of the switch regions conveys information about the identity of a particular Rab GTPase as well as the state of the bound nucleotide.     

Regulation of autophagy by the Rab GTPase network

Autophagy (macroautophagy) is a highly conserved intracellular and lysosome-dependent degradation process in which autophagic substrates are enclosed and degraded by a double-membrane vesicular structure in a continuous and dynamic vesicle transport process. The Rab protein is a small GTPase that belongs to the Ras-like GTPase superfamily and regulates the vesicle traffic process. Numerous Rab proteins have been shown to be involved in various stages of autophagy. Rab1, Rab5, Rab7, Rab9A, Rab11, Rab23, Rab32, and Rab33B participate in autophagosome formation, whereas Rab9 is required in non-canonical autophagy. Rab7, Rab8B, and Rab24 have a key role in autophagosome maturation. Rab8A and Rab25 are also involved in autophagy, but their role is unknown. Here, we summarize new findings regarding the involvement of Rabs in autophagy and provide insights regarding future research on the mechanisms of autophagy regulation.     

Giantin interacts with both the small GTPase Rab6 and Rab1

The interaction of small GTPases of the Rab family and coiled coil proteins of the golgin family has been reported for example for the Rab1 GTPase and p115, GM130 and Giantin. We now show that Rab6A, a GTPase that controls retrograde trafficking within the Golgi back to the endoplasmic reticulum is also able to bind to Giantin in vivo and in vitro pointing to an interesting complex formation between Giantin and two different Rab GTPases. In Saccharomyces cerevisiae a genetic interaction between Ypt1 and Ypt6 has already been demonstrated, but in this paper we were able to describe that the mammalian Rab GTPases are able to interact on the same golgin protein, Giantin.     

Rab6 interacts with the mint3 adaptor protein

The Rab6 GTPase regulates a retrograde transport route connecting endosomes and the endoplasmic reticulum (ER) via the Golgi apparatus. Recently it was shown that active (GTP-loaded) Rab6A regulates intracellular processing of the amyloid precursor protein (APP). To characterize the role of Rab6A in APP trafficking and to identify effector proteins of the active Rab6A protein, we screened a human placenta cDNA library using the yeast two-hybrid system. We isolated an interacting cDNA clone encoding part of the adaptor protein mint3. The interaction between Rab6A and mint3 is GTP-dependent and requires the complete phosphotyrosine-binding (PTB) domain of the mint protein, which also mediates the association with APP. By confocal microscopy we show that Rab6A, mint3 and APP co-localize at Golgi membranes in HeLa cells. Density gradient centrifugation of cytosolic extracts confirms a common distribution of these three proteins. Our data suggest that mint3 links Rab6A to APP traffic.     

The Hsp90 chaperone complex regulates GDI-dependent Rab recycling

Rab GTPase regulated hubs provide a framework for an integrated coding system, the membrome network, that controls the dynamics of the specialized exocytic and endocytic membrane architectures found in eukaryotic cells. Herein, we report that Rab recycling in the early exocytic pathways involves the heat-shock protein (Hsp)90 chaperone system. We find that Hsp90 forms a complex with guanine nucleotide dissociation inhibitor (GDI) to direct recycling of the client substrate Rab1 required for endoplasmic reticulum (ER)-to-Golgi transport. ER-to-Golgi traffic is inhibited by the Hsp90-specific inhibitors geldanamycin (GA), 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), and radicicol. Hsp90 activity is required to form a functional GDI complex to retrieve Rab1 from the membrane. Moreover, we find that Hsp90 is essential for Rab1-dependent Golgi assembly. The observation that the highly divergent Rab GTPases Rab1 involved in ER-to-Golgi transport and Rab3A involved in synaptic vesicle fusion require Hsp90 for retrieval from membranes lead us to now propose that the Hsp90 chaperone system may function as a general regulator for Rab GTPase recycling in exocytic and endocytic trafficking pathways involved in cell signaling and proliferation.     

A Highlights from MBoC Selection: Identification and characterization of multiple novel Rab–myosin Va interactions

Myosin Va is a widely expressed actin-based motor protein that binds members of the Rab GTPase family (3A, 8A, 10, 11A, 27A) and is implicated in many intracellular trafficking processes. To our knowledge, myosin Va has not been tested in a systematic screen for interactions with the entire Rab GTPase family. To that end, we report a yeast two-hybrid screen of all human Rabs for myosin Va-binding ability and reveal 10 novel interactions (3B, 3C, 3D, 6A, 6A′, 6B, 11B, 14, 25, 39B), which include interactions with three new Rab subfamilies (Rab6, Rab14, Rab39B). Of interest, myosin Va interacts with only a subset of the Rabs associated with the endocytic recycling and post-Golgi secretory systems. We demonstrate that myosin Va has three distinct Rab-binding domains on disparate regions of the motor (central stalk, an alternatively spliced exon, and the globular tail). Although the total pool of myosin Va is shared by several Rabs, Rab10 and Rab11 appear to be the major determinants of its recruitment to intracellular membranes. We also present evidence that myosin Va is necessary for maintaining a peripheral distribution of Rab11- and Rab14-positive endosomes.     

Characterizing the interaction between the Rab6 GTPase and Mint3 via flow cytometry based FRET analysis

In extension to previously applied techniques like yeast two-hybrid and GST pull-down assays, we successfully established a FACS-based FRET analysis to investigate the interaction of the Mint3 adaptor protein and the small Rab GTPase Rab6A in living mammalian cells. A Mint3 mutant containing only the PTB domain (Mint3Δ6) is able to interact with the constitutively active form of Rab6A. Mint3Δ4, a mutant lacking part of the PTB domain was unable to interact with Rab6A in GST pull-down analysis and did not produce FRET signals, when co-expressed with active Rab6A.We demonstrate that this FACS-based FRET analysis is a suitable method for interaction studies between two proteins in living cells.