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Intermediates in the Guanine Nucleotide Exchange Reaction of Rab8 Protein Catalyzed by Guanine Nucleotide Exchange Factors Rabin8 and GRAB*

  • Author Footnotes
    1 Both authors contributed equally to this work.
    ,
    Author Footnotes
    2 Present address: Inst. for Molecular Bioscience, The University of Queensland, St. Lucia 4072, Australia.
    Zhong Guo
    Footnotes
    1 Both authors contributed equally to this work.
    2 Present address: Inst. for Molecular Bioscience, The University of Queensland, St. Lucia 4072, Australia.
    Affiliations
    From the Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany,
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  • Author Footnotes
    1 Both authors contributed equally to this work.
    ,
    Author Footnotes
    3 Supported by National Natural Science Foundation of China Grant 31200554.
    Xiaomin Hou
    Footnotes
    1 Both authors contributed equally to this work.
    3 Supported by National Natural Science Foundation of China Grant 31200554.
    Affiliations
    From the Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany,

    the College of Life Science, Qingdao Agricultural University, Qingdao 266109, China
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  • Roger S. Goody
    Correspondence
    To whom correspondence may be addressed: Dept. of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany. Tel.: 49-231-133-2300
    Affiliations
    From the Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany,
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  • Aymelt Itzen
    Correspondence
    To whom correspondence may be addressed: Center for Integrated Protein Science Munich, Chemistry Dept., Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany. Tel.: 49-89-289-13343
    Affiliations
    From the Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany,

    the Center for Integrated Protein Science Munich, Chemistry Department, Technische Universität München, 85747 Garching, Germany
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  • Author Footnotes
    * This work was supported in part by German Research Foundation (DFG) Sonderforschungsbereich SFB1035 (Projekt B05) and SFB642 (Projekt A4).
    This article contains supplemental Figs. S1 and S2 and Table S1.
    1 Both authors contributed equally to this work.
    2 Present address: Inst. for Molecular Bioscience, The University of Queensland, St. Lucia 4072, Australia.
    3 Supported by National Natural Science Foundation of China Grant 31200554.
Open AccessPublished:September 26, 2013DOI:https://doi.org/10.1074/jbc.M113.498329
      Small G-proteins of the Ras superfamily control the temporal and spatial coordination of intracellular signaling networks by acting as molecular on/off switches. Guanine nucleotide exchange factors (GEFs) regulate the activation of these G-proteins through catalytic replacement of GDP by GTP. During nucleotide exchange, three distinct substrate·enzyme complexes occur: a ternary complex with GDP at the start of the reaction (G-protein·GEF·GDP), an intermediary nucleotide-free binary complex (G-protein·GEF), and a ternary GTP complex after productive G-protein activation (G-protein·GEF·GTP). Here, we show structural snapshots of the full nucleotide exchange reaction sequence together with the G-protein substrates and products using Rabin8/GRAB (GEF) and Rab8 (G-protein) as a model system. Together with a thorough enzymatic characterization, our data provide a detailed view into the mechanism of Rabin8/GRAB-mediated nucleotide exchange.
      Background: The GEFs Rabin8 and GRAB are activators of the vesicular trafficking regulator Rab8.
      Results: The catalytic mechanism of Rabin8/GRAB in Rab8 has been elucidated in biophysical and structural detail.
      Conclusion: Rabin8 and GRAB are catalytically moderately efficient enzymes and act by disturbing Mg2+ binding and Rab8-guanine base interactions.
      Significance: Obtaining snapshots of the nucleotide exchange reaction is crucial to understanding the mechanism of Rab GEFs.

      Introduction

      One of the hallmarks of eukaryotic cells is the intracellular movement of vesicles that transport material and allow communication between cellular compartments. The spatial and temporal regulation of vesicular trafficking is achieved by proteins of the Rab subfamily of small GTPases (
      • Cherfils J.
      • Zeghouf M.
      Regulation of small GTPases by GEFs, GAPs, and GDIs.
      ,
      • Hutagalung A.H.
      • Novick P.J.
      Role of Rab GTPases in membrane traffic and cell physiology.
      ). Rab proteins are molecular switches and cycle between inactive GDP-bound and active GTP-bound states. When inactive, Rab proteins exist in the cytosol in complex with the GDP dissociation inhibitor but are localized to a distinct membrane when in the active state. To exert their function, Rab proteins need to be activated by a process requiring guanine nucleotide exchange factors (GEFs).
      The abbreviations used are: GEF, guanine nucleotide exchange factor; GppNHp, 5′-guanylyl imidodiphosphate; mant, N-methylanthraniloyl.
      These enzymes accelerate GDP release from and allow the binding of GTP to a Rab protein. Rab proteins can interact with effector proteins that preferentially bind the active GTP but not the GDP state. GTPase-activating proteins stimulate the very low intrinsic GTPase activity of Rab proteins and thus convert them back into the inactive form.
      The Rab subfamily consists of ∼60 members in humans, and each family member has a specific intracellular localization (
      • Hutagalung A.H.
      • Novick P.J.
      Role of Rab GTPases in membrane traffic and cell physiology.
      ). The correct activation of a certain Rab requires the action of a cognate Rab GEF at the proper location and at the appropriate time (
      • Blümer J.
      • Rey J.
      • Dehmelt L.
      • Mazel T.
      • Wu Y.W.
      • Bastiaens P.
      • Goody R.S.
      • Itzen A.
      RabGEFs are a major determinant for specific Rab membrane targeting.
      ). Consequently, GEFs have evolved to have mechanisms that guarantee their correct membrane targeting as well as the specific recognition of one distinct Rab protein over structurally and sequentially similar family members.
      The Rab protein Rab8 is involved in events such as the delivery of secretory vesicles to the plasma membrane and polarized membrane transport in epithelial cells (
      • Peränen J.
      • Auvinen P.
      • Virta H.
      • Wepf R.
      • Simons K.
      Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts.
      ,
      • Hattula K.
      • Furuhjelm J.
      • Arffman A.
      • Peränen J.
      A Rab8-specific GDP/GTP exchange factor is involved in actin remodeling and polarized membrane transport.
      ). Rab8 also regulates cell shape, and interaction with its GEF Rabin8 appears to be crucial to this function (
      • Hattula K.
      • Furuhjelm J.
      • Tikkanen J.
      • Tanhuanpää K.
      • Laakkonen P.
      • Peränen J.
      Characterization of the Rab8-specific membrane traffic route linked to protrusion formation.
      ). Rabin8 and Rab8 appear to be important in cilium formation by acting in concert with the Bardet-Biedl syndrome complex (
      • Nachury M.V.
      • Loktev A.V.
      • Zhang Q.
      • Westlake C.J.
      • Peränen J.
      • Merdes A.
      • Slusarski D.C.
      • Scheller R.H.
      • Bazan J.F.
      • Sheffield V.C.
      • Jackson P.K.
      A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis.
      ,
      • Yoshimura S.
      • Egerer J.
      • Fuchs E.
      • Haas A.K.
      • Barr F.A.
      Functional dissection of Rab GTPases involved in primary cilium formation.
      ,
      • Peränen J.
      Rab8 GTPase as a regulator of cell shape.
      ). Rabin8 is a 460-amino acid protein that consists of several domains, only two of which are functionally characterized (
      • Hattula K.
      • Furuhjelm J.
      • Arffman A.
      • Peränen J.
      A Rab8-specific GDP/GTP exchange factor is involved in actin remodeling and polarized membrane transport.
      ,
      • Knödler A.
      • Feng S.
      • Zhang J.
      • Zhang X.
      • Das A.
      • Peränen J.
      • Guo W.
      Coordination of Rab8 and Rab11 in primary ciliogenesis.
      ,
      • Westlake C.J.
      • Baye L.M.
      • Nachury M.V.
      • Wright K.J.
      • Ervin K.E.
      • Phu L.
      • Chalouni C.
      • Beck J.S.
      • Kirkpatrick D.S.
      • Slusarski D.C.
      • Sheffield V.C.
      • Scheller R.H.
      • Jackson P.K.
      Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome.
      ). Rabin8 contains a central Sec2 domain with GEF activity toward Rab8 (
      • Hattula K.
      • Furuhjelm J.
      • Arffman A.
      • Peränen J.
      A Rab8-specific GDP/GTP exchange factor is involved in actin remodeling and polarized membrane transport.
      ). Amino acid sequence and structure comparison with the yeast homolog Sec2 predicts that the central domain of Rabin8 consists of a homodimeric parallel coiled coil. Rabin8 is thought to be recruited to its target location by active Rab11, which interacts with a C-terminal Rab11 effector domain (
      • Knödler A.
      • Feng S.
      • Zhang J.
      • Zhang X.
      • Das A.
      • Peränen J.
      • Guo W.
      Coordination of Rab8 and Rab11 in primary ciliogenesis.
      ). Another factor possessing a Sec2-like GEF domain is the 382-amino acid protein GRAB. Despite a high sequence homology of the Sec2 domain of GRAB to Rabin8, it has previously been reported that GRAB is a GEF for Rab3A rather than Rab8 (
      • Luo H.R.
      • Saiardi A.
      • Nagata E.
      • Ye K.
      • Yu H.
      • Jung T.S.
      • Luo X.
      • Jain S.
      • Sawa A.
      • Snyder S.H.
      GRAB: a physiologic guanine nucleotide exchange factor for Rab3A, which interacts with inositol hexakisphosphate kinase.
      ). However, a recent study that analyzed the activity profiles of various Rab GEFs (with a focus on the DENN domain family, which is structurally unrelated to GRAB) indicated that GRAB has GEF activity toward Rab8 but not Rab3 (
      • Yoshimura S.
      • Gerondopoulos A.
      • Linford A.
      • Rigden D.J.
      • Barr F.A.
      Family-wide characterization of the DENN domain Rab GDP-GTP exchange factors.
      ).
      The mode of action of GTPases involved in signal transduction or regulation includes activation of GDP release catalyzed by a specific GEF in almost all cases known. The basic mechanistic feature is the weakening of the otherwise very tight binding of GDP (Kd values in the nanomolar to picomolar range) by interaction with GEFs, which also bind with similarly high affinity to their cognate GTPases (
      • Goody R.S.
      • Hofmann-Goody W.
      Exchange factors, effectors, GAPs and motor proteins: common thermodynamic and kinetic principles for different functions.
      ). The effect is both thermodynamic and kinetic, implying the formation of a ternary complex between GEF, GTPase, and GDP, with a dramatic reduction in the affinities of both GEF and GDP in the ternary complex in comparison with the respective binary complexes. Several GTPase-GEF interactions have been examined thoroughly at the kinetic level, and a large number of GTPase·GEF complexes have been characterized structurally.
      In this work, we have examined the interaction of the Ras superfamily protein Rab8 with two structurally highly similar GEF molecules (Rabin8 and GRAB) by kinetic and structural methods. GRAB is a GEF for Rab8 with almost identical structural properties to Rabin8. The work presented led to the identification of several intermediates in the overall GDP/GTP exchange of Rab8 in the presence of Rabin8.

      DISCUSSION

      In this work, we have investigated the GEF reactions of Rabin8 and GRAB with their substrate Rab8 in biochemical and structural detail. Kinetic analysis of the GEF Sec2 domains of GRAB and Rabin8 revealed that they possess a moderately efficient catalytic activity. Unexpectedly, the affinity of the Rab·GEF complex for nucleotides is relatively high (KD4 = 0.26 μm for GDP), allowing the generation of ternary Rab8·Rabin8/GRAB·nucleotide complexes with GDP and GTP. The structures of these complexes allow us to describe the nucleotide exchange in detail at the molecular level.
      The mechanism of GDP displacement in the Rab8/Rabin8 system can be described as a disturbance of the structure of the nucleotide-binding regions of Rab8, in particular switch I and switch II. In previously determined structures of GTPase·GEF complexes, the direct interaction of the GEF with switch II appears to be a universal feature. Contacts with switch I occur in many (but not all) complexes, whereas contacts with the P-loop are less common but are seen in some instances. In Rab8·Rabin8, interaction of the C-terminal part of switch II leads to the formation of an α-helix and displacement of Asp-63 from its position near to the nucleotide-binding site, where it is involved in an indirect (via a water molecule) interaction with Mg2+ in the Rab8·GDP and Rab8·GppNHp structures. This interaction is highly conserved in the Ras superfamily. In the absence of GEFs, the highly conserved Gly-66 residue interacts via its backbone NH with the γ-phosphate of GTP but not GDP. In the GDP displacement mechanism, the disturbance of the Mg2+ coordination presumably contributes to destabilization of the metal ion at this site. Further destabilization arises from structural changes induced in switch I, leading to displacement of Mg2+ by Ile-38. Similar effects are seen in other GTPase·GEF complexes, with the residue displacing Mg2+ coming either from the GTPase itself (
      • Boriack-Sjodin P.A.
      • Margarit S.M.
      • Bar-Sagi D.
      • Kuriyan J.
      The structural basis of the activation of Ras by Sos.
      ,
      • Worthylake D.K.
      • Rossman K.L.
      • Sondek J.
      Crystal structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1.
      ) or from the GEF (
      • Béraud-Dufour S.
      • Robineau S.
      • Chardin P.
      • Paris S.
      • Chabre M.
      • Cherfils J.
      • Antonny B.
      A glutamic finger in the guanine nucleotide exchange factor ARNO displaces Mg2+ and the β-phosphate to destabilize GDP on ARF1.
      ,
      • Uejima T.
      • Ihara K.
      • Goh T.
      • Ito E.
      • Sunada M.
      • Ueda T.
      • Nakano A.
      • Wakatsuki S.
      GDP-bound and nucleotide-free intermediates of the guanine nucleotide exchange in the Rab5·Vps9 system.
      ). As determined in earlier work on Rab7, removal of Mg2+ leads to an acceleration of a factor of ∼250 for GDP dissociation (
      • Simon I.
      • Zerial M.
      • Goody R.S.
      Kinetics of interaction of Rab5 and Rab7 with nucleotides and magnesium ions.
      ).
      Another universally conserved effect seen upon interaction of GEFs with members of the Ras superfamily is the disturbance of the interaction of a highly conserved phenylalanine in switch I (Phe-33 in Rab8) with the guanine base. In the case of Ras, it was shown that mutation of this residue to isoleucine results in an ∼100-fold increase in the rate constant for GDP dissociation (
      • Reinstein J.
      • Schlichting I.
      • Frech M.
      • Goody R.S.
      • Wittinghofer A.
      p21 with a phenylalanine 28 → leucine mutation reacts normally with the GTPase activating protein GAP but nevertheless has transforming properties.
      ). Assuming an additive contribution of both of these mechanisms (i.e. removal of Mg2+ and disruption of the Phe-33 interaction) to the dissociation rate of GDP, this would imply an acceleration of ∼2.5 × 104 for Rab8·Rabin8·GDP relative to Rab8·GDP. The factor determined (k″−1/k−4 in Scheme 1) is actually smaller (∼1.5 × 103), which could indicate that there is rate limitation for Mg2+ dissociation or Phe-33 movement. Rabin8 has a similar catalytic efficiency as other GEFs, with GDP/GTP exchange accelerations in the range of ∼102–104-fold, but some GEFs are considerably faster. Among these are Cdc25 (for Ras; acceleration factor of ∼2 × 105), DrrA from L. pneumophila (for Rab1b; ∼8 × 105-fold), and RCC1 (for Ran; ∼1.3 × 106-fold) (
      • Lenzen C.
      • Cool R.H.
      • Prinz H.
      • Kuhlmann J.
      • Wittinghofer A.
      Kinetic analysis by fluorescence of the interaction between Ras and the catalytic domain of the guanine nucleotide exchange factor Cdc25Mm.
      ,
      • Schoebel S.
      • Oesterlin L.K.
      • Blankenfeldt W.
      • Goody R.S.
      • Itzen A.
      RabGDI displacement by DrrA from Legionella is a consequence of its guanine nucleotide exchange activity.
      ,
      • Klebe C.
      • Prinz H.
      • Wittinghofer A.
      • Goody R.S.
      The kinetic mechanism of Ran-nucleotide exchange catalyzed by RCC1.
      ).
      It has recently been pointed out that in many GTPase·GEF complexes of the Ras superfamily, the conserved lysine of the P-loop (Lys-21 in Rab8 and Lys-16 in H-Ras), which interacts with the β-phosphate of GDP or GTP, is intermediately stabilized by a conserved glutamate in the G3 motif at the beginning of switch II (DxxGQE motif; Glu-62 in Ras) (
      • Gasper R.
      • Thomas C.
      • Ahmadian M.R.
      • Wittinghofer A.
      The role of the conserved switch II glutamate in guanine nucleotide exchange factor-mediated nucleotide exchange of GTP-binding proteins.
      ). In a number of cases, substitution of this glutamate by alanine results in significant impairment of GEF activity. The corresponding residue is conserved in Rab molecules but does not appear to be involved in such an interaction and is not essential for the exchange reaction (
      • Gasper R.
      • Thomas C.
      • Ahmadian M.R.
      • Wittinghofer A.
      The role of the conserved switch II glutamate in guanine nucleotide exchange factor-mediated nucleotide exchange of GTP-binding proteins.
      ). In this work, the corresponding Rab8 P-loop lysine (Lys-21) interacts with GTP, GDP, or sulfate in the crystal structures of Rab8·Rabin8. Because sulfate is bound in the nucleotide-free state, we do not in fact have a structure corresponding to the genuine nucleotide-free situation, so we cannot make a definitive statement concerning the possible formation of an interaction of Lys-21 and Glu-68 in the nucleotide-free state. Although we cannot present a bona fide nucleotide-free Rab8·Rabin8 structure (the sulfate presumably mimics the β-phosphate position), the ϵ-amino group of Lys-21 is close to the carboxylate of Asp-63 of the DxxGQE sequence and could thus interact with Lys-21 in a hypothetical Rab8·Rabin8 complex devoid of nucleotides. This interaction (i.e. lysine of the P-loop with aspartate of the DxxGQE motif) is seen in at least two other Rab·GEF complexes (Rabex5·Rab21 and DrrA·Rab1b), as well as in the Ran·RCC1 complex. It therefore appears that nucleotide-free states of GTPase·GEF complexes are stabilized in part by interactions of the P-loop lysine with the glutamate of the DxxGQE motif, with the aspartate, or, in some cases, with both acidic residues.
      Rabin8 and GRAB are specific GEFs for Rab8, in contrast to the rather promiscuous protein MSS4, which shows GEF activity toward Rab1, Rab3, Rab10, and Rab13 in addition to Rab8 (
      • Itzen A.
      • Pylypenko O.
      • Goody R.S.
      • Alexandrov K.
      • Rak A.
      Nucleotide exchange via local protein unfolding–structure of Rab8 in complex with MSS4.
      ,
      • Wixler V.
      • Wixler L.
      • Altenfeld A.
      • Ludwig S.
      • Goody R.S.
      • Itzen A.
      Identification and characterisation of novel Mss4-binding Rab GTPases.
      ,
      • Burton J.L.
      • Burns M.E.
      • Gatti E.
      • Augustine G.J.
      • De Camilli P.
      Specific interactions of Mss4 with members of the Rab GTPase subfamily.
      ). MSS4 is a catalytically inefficient GEF with a kcat/Km value about four times lower than that of Rabin8/GRAB. More importantly, the Rab8·MSS4 complex displays a very small second-order rate constant of GTP reassociation (
      • Itzen A.
      • Pylypenko O.
      • Goody R.S.
      • Alexandrov K.
      • Rak A.
      Nucleotide exchange via local protein unfolding–structure of Rab8 in complex with MSS4.
      ); therefore, the Rab8·MSS4 complex remains in the nucleotide-free form for a considerably longer time than other GEFs, such as Rabin8/GRAB. It is believed that the weak GEF properties of MSS4 are a result of its enzyme mechanism, in which nucleotide release is induced by a local protein-unfolding reaction of Rab8 (
      • Itzen A.
      • Pylypenko O.
      • Goody R.S.
      • Alexandrov K.
      • Rak A.
      Nucleotide exchange via local protein unfolding–structure of Rab8 in complex with MSS4.
      ): in contrast to Rabin8/GRAB, MSS4 unfolds the entire switch I region together with α-helix α1 of Rab8. This mechanism requires refolding of the nucleotide-binding pocket prior to GTP rebinding, and thus, nucleotide association is impaired. However, Sec2 domain GEFs (such as Rabin8 and GRAB) act differently from MSS4 because they stabilize the nucleotide-binding pocket by keeping switch I, switch II, and the P-loop in structurally defined confirmations that allow unimpaired rebinding of nucleotides (
      • Sato Y.
      • Fukai S.
      • Ishitani R.
      • Nureki O.
      Crystal structure of the Sec4p·Sec2p complex in the nucleotide exchanging intermediate state.
      ,
      • Dong G.
      • Medkova M.
      • Novick P.
      • Reinisch K.M.
      A catalytic coiled coil: structural insights into the activation of the Rab GTPase Sec4p by Sec2p.
      ).
      The work presented describes the structures of several intermediates in the exchange mechanism of Rabin8/GRAB with respect to Rab8. What is still missing is the structure of an intermediate in which Mg2+ is bound. Such a structure has been described recently for the GEF·GTPase pair DOCK9·Cdc42 in the presence of GTP (
      • Yang J.
      • Zhang Z.
      • Roe S.M.
      • Marshall C.J.
      • Barford D.
      Activation of Rho GTPases by DOCK exchange factors is mediated by a nucleotide sensor.
      ). Here, the amino acid homologous to Ile-38 in Rab8 (a valine from the DOCK9 molecule) is displaced from its Mg2+-occluding position in the presence of Mg2+ and GTP, but not in the presence of Mg2+ and GDP. The authors interpreted this as a nucleotide sensor, leading to an alleged preference of the GEF for the GDP state of the GTPase over the GTP form and therefore allowing an unidirectional nucleotide exchange reaction. However, a similar effect is not observed in our structure, and the mode of nucleotide binding to Rab8·Rabin8/GRAB is identical for GDP and GTP. This is in keeping with the observation that there is no general preference for GEF interaction with the GDP or GTP form of GTPases, as required on theoretical grounds (
      • Goody R.S.
      • Hofmann-Goody W.
      Exchange factors, effectors, GAPs and motor proteins: common thermodynamic and kinetic principles for different functions.
      ).

      Acknowledgments

      We thank Nathalie Bleimling for expert support in protein production. We thank the x-ray community of Max Planck Institute of Molecular Physiology Dortmund for help with data collection and the staff of beamline X10SA at the Swiss Light Source (Paul Scherrer Institute) for generous access to the facilities. We thank Prof. Wulf Blankenfeldt for invaluable help in structure determination.

      REFERENCES

        • Cherfils J.
        • Zeghouf M.
        Regulation of small GTPases by GEFs, GAPs, and GDIs.
        Physiol. Rev. 2013; 93: 269-309
        • Hutagalung A.H.
        • Novick P.J.
        Role of Rab GTPases in membrane traffic and cell physiology.
        Physiol. Rev. 2011; 91: 119-149
        • Blümer J.
        • Rey J.
        • Dehmelt L.
        • Mazel T.
        • Wu Y.W.
        • Bastiaens P.
        • Goody R.S.
        • Itzen A.
        RabGEFs are a major determinant for specific Rab membrane targeting.
        J. Cell Biol. 2013; 200: 287-300
        • Peränen J.
        • Auvinen P.
        • Virta H.
        • Wepf R.
        • Simons K.
        Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts.
        J. Cell Biol. 1996; 135: 153-167
        • Hattula K.
        • Furuhjelm J.
        • Arffman A.
        • Peränen J.
        A Rab8-specific GDP/GTP exchange factor is involved in actin remodeling and polarized membrane transport.
        Mol. Biol. Cell. 2002; 13: 3268-3280
        • Hattula K.
        • Furuhjelm J.
        • Tikkanen J.
        • Tanhuanpää K.
        • Laakkonen P.
        • Peränen J.
        Characterization of the Rab8-specific membrane traffic route linked to protrusion formation.
        J. Cell Sci. 2006; 119: 4866-4877
        • Nachury M.V.
        • Loktev A.V.
        • Zhang Q.
        • Westlake C.J.
        • Peränen J.
        • Merdes A.
        • Slusarski D.C.
        • Scheller R.H.
        • Bazan J.F.
        • Sheffield V.C.
        • Jackson P.K.
        A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis.
        Cell. 2007; 129: 1201-1213
        • Yoshimura S.
        • Egerer J.
        • Fuchs E.
        • Haas A.K.
        • Barr F.A.
        Functional dissection of Rab GTPases involved in primary cilium formation.
        J. Cell Biol. 2007; 178: 363-369
        • Peränen J.
        Rab8 GTPase as a regulator of cell shape.
        Cytoskeleton. 2011; 68: 527-539
        • Knödler A.
        • Feng S.
        • Zhang J.
        • Zhang X.
        • Das A.
        • Peränen J.
        • Guo W.
        Coordination of Rab8 and Rab11 in primary ciliogenesis.
        Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 6346-6351
        • Westlake C.J.
        • Baye L.M.
        • Nachury M.V.
        • Wright K.J.
        • Ervin K.E.
        • Phu L.
        • Chalouni C.
        • Beck J.S.
        • Kirkpatrick D.S.
        • Slusarski D.C.
        • Sheffield V.C.
        • Scheller R.H.
        • Jackson P.K.
        Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome.
        Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 2759-2764
        • Luo H.R.
        • Saiardi A.
        • Nagata E.
        • Ye K.
        • Yu H.
        • Jung T.S.
        • Luo X.
        • Jain S.
        • Sawa A.
        • Snyder S.H.
        GRAB: a physiologic guanine nucleotide exchange factor for Rab3A, which interacts with inositol hexakisphosphate kinase.
        Neuron. 2001; 31: 439-451
        • Yoshimura S.
        • Gerondopoulos A.
        • Linford A.
        • Rigden D.J.
        • Barr F.A.
        Family-wide characterization of the DENN domain Rab GDP-GTP exchange factors.
        J. Cell Biol. 2010; 191: 367-381
        • Goody R.S.
        • Hofmann-Goody W.
        Exchange factors, effectors, GAPs and motor proteins: common thermodynamic and kinetic principles for different functions.
        Eur. Biophys. J. 2002; 31: 268-274
        • Bleimling N.
        • Alexandrov K.
        • Goody R.
        • Itzen A.
        Chaperone-assisted production of active human Rab8A GTPase in Escherichia coli.
        Protein Expr. Purif. 2009; 65: 190-195
        • Mihai Gazdag E.
        • Streller A.
        • Haneburger I.
        • Hilbi H.
        • Vetter I.R.
        • Goody R.S.
        • Itzen A.
        Mechanism of Rab1b deactivation by the Legionella pneumophila GAP LepB.
        EMBO Rep. 2013; 14: 199-205
        • Berrow N.S.
        • Alderton D.
        • Sainsbury S.
        • Nettleship J.
        • Assenberg R.
        • Rahman N.
        • Stuart D.I.
        • Owens R.J.
        A versatile ligation-independent cloning method suitable for high-throughput expression screening applications.
        Nucleic Acids Res. 2007; 35: e45
        • Hou X.
        • Hagemann N.
        • Schoebel S.
        • Blankenfeldt W.
        • Goody R.S.
        • Erdmann K.S.
        • Itzen A.
        A structural basis for Lowe syndrome caused by mutations in the Rab-binding domain of OCRL1.
        EMBO J. 2011; 30: 1659-1670
        • Müller M.P.
        • Peters H.
        • Blümer J.
        • Blankenfeldt W.
        • Goody R.S.
        • Itzen A.
        The Legionella effector protein DrrA AMPylates the membrane traffic regulator Rab1b.
        Science. 2010; 329: 946-949
        • Kabsch W.
        Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants.
        J. Appl. Crystallogr. 1993; 26: 795-800
        • McCoy A.J.
        • Grosse-Kunstleve R.W.
        • Storoni L.C.
        • Read R.J.
        Likelihood-enhanced fast translation functions.
        Acta Crystallogr. D Biol. Crystallogr. 2005; 61: 458-464
        • Murshudov G.N.
        • Vagin A.A.
        • Lebedev A.
        • Wilson K.S.
        • Dodson E.J.
        Efficient anisotropic refinement of macromolecular structures using FFT.
        Acta Crystallogr. D Biol. Crystallogr. 1999; 55: 247-255
        • Emsley P.
        • Lohkamp B.
        • Scott W.G.
        • Cowtan K.
        Features and development of Coot.
        Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 486-501
        • Itzen A.
        • Rak A.
        • Goody R.S.
        Sec2 is a highly efficient exchange factor for the Rab protein Sec4.
        J. Mol. Biol. 2007; 365: 1359-1367
        • Itzen A.
        • Pylypenko O.
        • Goody R.S.
        • Alexandrov K.
        • Rak A.
        Nucleotide exchange via local protein unfolding–structure of Rab8 in complex with MSS4.
        EMBO J. 2006; 25: 1445-1455
        • Lenzen C.
        • Cool R.H.
        • Prinz H.
        • Kuhlmann J.
        • Wittinghofer A.
        Kinetic analysis by fluorescence of the interaction between Ras and the catalytic domain of the guanine nucleotide exchange factor Cdc25Mm.
        Biochemistry. 1998; 37: 7420-7430
        • Schoebel S.
        • Oesterlin L.K.
        • Blankenfeldt W.
        • Goody R.S.
        • Itzen A.
        RabGDI displacement by DrrA from Legionella is a consequence of its guanine nucleotide exchange activity.
        Mol. Cell. 2009; 36: 1060-1072
        • Sato Y.
        • Fukai S.
        • Ishitani R.
        • Nureki O.
        Crystal structure of the Sec4p·Sec2p complex in the nucleotide exchanging intermediate state.
        Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 8305-8310
        • Buchwald G.
        • Friebel A.
        • Galán J.E.
        • Hardt W.D.
        • Wittinghofer A.
        • Scheffzek K.
        Structural basis for the reversible activation of a Rho protein by the bacterial toxin SopE.
        EMBO J. 2002; 21: 3286-3295
        • Coureux P.D.
        • Wells A.L.
        • Ménétrey J.
        • Yengo C.M.
        • Morris C.A.
        • Sweeney H.L.
        • Houdusse A.
        A structural state of the myosin V motor without bound nucleotide.
        Nature. 2003; 425: 419-423
        • Reinstein J.
        • Schlichting I.
        • Frech M.
        • Goody R.S.
        • Wittinghofer A.
        p21 with a phenylalanine 28 → leucine mutation reacts normally with the GTPase activating protein GAP but nevertheless has transforming properties.
        J. Biol. Chem. 1991; 266: 17700-17706
        • Boriack-Sjodin P.A.
        • Margarit S.M.
        • Bar-Sagi D.
        • Kuriyan J.
        The structural basis of the activation of Ras by Sos.
        Nature. 1998; 394: 337-343
        • Worthylake D.K.
        • Rossman K.L.
        • Sondek J.
        Crystal structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1.
        Nature. 2000; 408: 682-688
        • Béraud-Dufour S.
        • Robineau S.
        • Chardin P.
        • Paris S.
        • Chabre M.
        • Cherfils J.
        • Antonny B.
        A glutamic finger in the guanine nucleotide exchange factor ARNO displaces Mg2+ and the β-phosphate to destabilize GDP on ARF1.
        EMBO J. 1998; 17: 3651-3659
        • Uejima T.
        • Ihara K.
        • Goh T.
        • Ito E.
        • Sunada M.
        • Ueda T.
        • Nakano A.
        • Wakatsuki S.
        GDP-bound and nucleotide-free intermediates of the guanine nucleotide exchange in the Rab5·Vps9 system.
        J. Biol. Chem. 2010; 285: 36689-36697
        • Simon I.
        • Zerial M.
        • Goody R.S.
        Kinetics of interaction of Rab5 and Rab7 with nucleotides and magnesium ions.
        J. Biol. Chem. 1996; 271: 20470-20478
        • Klebe C.
        • Prinz H.
        • Wittinghofer A.
        • Goody R.S.
        The kinetic mechanism of Ran-nucleotide exchange catalyzed by RCC1.
        Biochemistry. 1995; 34: 12543-12552
        • Gasper R.
        • Thomas C.
        • Ahmadian M.R.
        • Wittinghofer A.
        The role of the conserved switch II glutamate in guanine nucleotide exchange factor-mediated nucleotide exchange of GTP-binding proteins.
        J. Mol. Biol. 2008; 379: 51-63
        • Wixler V.
        • Wixler L.
        • Altenfeld A.
        • Ludwig S.
        • Goody R.S.
        • Itzen A.
        Identification and characterisation of novel Mss4-binding Rab GTPases.
        Biol. Chem. 2011; 392: 239-248
        • Burton J.L.
        • Burns M.E.
        • Gatti E.
        • Augustine G.J.
        • De Camilli P.
        Specific interactions of Mss4 with members of the Rab GTPase subfamily.
        EMBO J. 1994; 13: 5547-5558
        • Dong G.
        • Medkova M.
        • Novick P.
        • Reinisch K.M.
        A catalytic coiled coil: structural insights into the activation of the Rab GTPase Sec4p by Sec2p.
        Mol. Cell. 2007; 25: 455-462
        • Yang J.
        • Zhang Z.
        • Roe S.M.
        • Marshall C.J.
        • Barford D.
        Activation of Rho GTPases by DOCK exchange factors is mediated by a nucleotide sensor.
        Science. 2009; 325: 1398-1402