Structural basis for Rab GTPase activation by VPS9 domain exchange factors

Delprato, Anna; Lambright, David G.

doi:10.1038/nsmb1232

Cited by 124 publications

(204 citation statements)

References 55 publications

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“…Note added in proof. While this article was in revision, a new paper was published and showed that a region downstream of the Vps9 domain exerted "autoinhibition" on the GEF activity of Rabex-5 and that this inhibition could be partially relieved by Rabaptin-5 in vitro (Delprato and Lambright, 2007). These results are consistent with our data in Figure 2.…”

Section: Discussionsupporting

confidence: 83%

Rabaptin-5-independent Membrane Targeting and Rab5 Activation by Rabex-5 in the Cell

Zhu

Liu

et al. 2007

MBoC

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Rabex-5 is a guanine nucleotide exchange factor (GEF) for Rab5. Here, we report the identification of a novel functional domain of Rabex-5 that is essential for its membrane targeting and Rab5 GEF activity in vivo. The data show that full-length Rabex-5 efficiently activates Rab5 in the cell. However, the GEF domain itself (residues 135-399) is inactive in this respect, despite its activity in vitro. Generation and characterization of a series of Rabex-5 constructs reveal that the GEF domain is unable to target to early endosomes and that a sequence N-terminal to the GEF domain can restore its early endosomal targeting and its ability to activate Rab5 in the cell. This region (residues 81-135) is termed membrane-binding motif, which together with the downstream helical bundle domain (residues 135-230) forms an early endosomal targeting (EET) domain necessary and sufficient for association with early endosomes. Furthermore, several active Rabex-5 constructs do not contain the Rabaptin-5-binding domain in the C-terminal region. Thus, Rabex-5 can target to early endosomes via the EET domain and activate Rab5 in a Rabaptin-5-independent manner in vivo. We discuss a model to reconcile these in vivo data with previous in vitro results on Rabex-5 function and its interaction with Rabaptin-5.

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Section: Discussionsupporting

confidence: 83%

Rabaptin-5-independent Membrane Targeting and Rab5 Activation by Rabex-5 in the Cell

Zhu

Liu

et al. 2007

MBoC

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“…Our findings suggest that the HVDs of individual Rab proteins play distinct roles in membrane targeting. In the case of Rab1 and Rab5, the HVD is not required for membrane targeting, probably because it is not involved in binding with potential targeting factors, including effectors and GEFs (12,(31)(32)(33). It serves rather as an anchoring chain that physically connects the functional GTPase domain with the membrane.…”

Section: Discussionmentioning

confidence: 99%

The role of the hypervariable C-terminal domain in Rab GTPases membrane targeting

Zhao

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Intracellular membrane trafficking requires correct and specific localization of Rab GTPases. The hypervariable C-terminal domain (HVD) of Rabs is posttranslationally modified by isoprenyl moieties that enable membrane association. A model asserting HVDdirected targeting has been contested in previous studies, but the role of the Rab HVD and the mechanism of Rab membrane targeting remain elusive. To elucidate the function of the HVD, we have substituted this region with an unnatural polyethylenglycol (PEG) linker by using oxime ligation. The PEGylated Rab proteins undergo normal prenylation, underlining the unique ability of the Rab prenylation machinery to process the Rab family with diverse C-terminal sequences. Through localization studies and functional analyses of semisynthetic PEGylated Rab1, Rab5, Rab7, and Rab35 proteins, we demonstrate that the role of the HVD of Rabs in membrane targeting is more complex than previously understood. The HVD of Rab1 and Rab5 is dispensable for membrane targeting and appears to function simply as a linker between the GTPase domain and the membrane. The N-terminal residues of the Rab7 HVD are important for late endosomal/lysosomal localization, apparently due to their involvement in interaction with the Rab7 effector Rabinteracting lysosomal protein. The C-terminal polybasic cluster of the Rab35 HVD is essential for plasma membrane (PM) targeting, presumably because of the electrostatic interaction with negatively charged lipids on the PM. Our findings suggest that Rab membrane targeting is dictated by a complex mechanism involving GEFs, GAPs, effectors, and C-terminal interaction with membranes to varying extents, and possibly other binding partners. Interacting with a complex network of Rab regulators and effectors, Rab GTPases regulate these processes through a spatiotemporally controlled GTPase cycle and their distribution in cells. The GTPase cycle is strictly regulated by guanine nucleotide exchange factors (GEFs) that mediate GDP/GTP exchange and by GTPase-activating proteins (GAPs) that accelerate the hydrolysis of GTP. Active (GTP-bound) Rab proteins associate with distinct intracellular compartments and direct vesicular transport by recruiting a multitude of Rab-specific effectors, including tethering complexes and motor proteins.Rab proteins are posttranslationally modified at the C terminus with prenyl groups that function as membrane anchors. Rab prenylation involves covalent attachment of the geranylgeranyl (C-20 isoprenyl) moiety to one or two C-terminal cysteine residues of the protein substrate via a stable thioether linkage (4). Unlike other protein prenyltransferases that recognize the Cterminal CaaX motif of protein substrates (e.g., Ras and Rho), Rab geranylgeranyl transferase (RabGGTase) does not recognize its protein substrates (Rab proteins) directly but requires the adaptor Rab escort protein (REP). Rab prenylation requires the formation of a ternary catalytic Rab:REP:RabGGTase complex (5-7). It remains elusive how the single Rab prenylation machin...

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“…As the GEF activities of these proteins might be regulated by conformation changes due to upstream signals, structures other than VPS9 domains might alter their GEF activities. Actually, the exchange activities of Rabex-5 and Varp are suppressed by autoinhibitory elements present in the C terminus of VPS9 domains, and the elimination of these regions resulted in GEF activation (18,30). Taking into consideration that downregulation of Gapex-5 impaired insulin-stimulated PKB/Akt activation (42), the GEF activity of Gapex-5 might be elevated by stimulators, including growth factors.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, they can be functionally categorized into the Rab5 subfamily (29). Recent reports show that Rab21 is activated by Varp and Rabex-5 (18,30) and that Rab31 is activated by , with each of these proteins containing a VPS9 domain. However, the activation mechanisms of Rab31 are poorly understood, and other VPS9 domain proteins might act as Rab31-GEFs in this paradigm.…”

mentioning

confidence: 99%

Characterization of RIN3 as a Guanine Nucleotide Exchange Factor for the Rab5 Subfamily GTPase Rab31

Kajiho

Sakurai

Minoda

et al. 2011

Journal of Biological Chemistry

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The small GTPase Rab5, which cycles between GDP-bound inactive and GTP-bound active forms, plays essential roles in membrane budding and trafficking in the early endocytic pathway. Rab5 is activated by various vacuolar protein sorting 9 (VPS9) domain-containing guanine nucleotide exchange factors. Rab21, Rab22, and Rab31 (members of the Rab5 subfamily) are also involved in the trafficking of early endosomes. Mechanisms controlling the activation Rab5 subfamily members remain unclear. RIN (Ras and Rab interactor) represents a family of multifunctional proteins that have a VPS9 domain in addition to Src homology 2 (SH2) and Ras association domains. We investigated whether RIN family members act as guanine nucleotide exchange factors (GEFs) for the Rab5 subfamily on biochemical and cell morphological levels. RIN3 stimulated the formation of GTP-bound Rab31 in cell-free and in cell GEF activity assays. RIN3 also formed enlarged vesicles and tubular structures, where it colocalized with Rab31 in HeLa cells. In contrast, RIN3 did not exhibit any apparent effects on Rab21. We also found that serine to alanine substitutions in the sequences between SH2 and RIN family homology domain of RIN3 specifically abolished its GEF action on Rab31 but not Rab5. We examined whether RIN3 affects localization of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is transported between trans-Golgi network and endocytic compartments. We found that RIN3 partially translocates CD-MPR from the trans-Golgi network to peripheral vesicles and that this is dependent on its Rab31-GEF activity. These results indicate that RIN3 specifically acts as a GEF for Rab31.The small GTPase Rab family plays pivotal roles in intracellular membrane trafficking. At present, Ͼ60 Rab GTPases have been identified, and they localize to distinct intracellular compartments that organize transport in specific organelles (1-3). The functional state of Rab GTPases depends on structural conformation, which is determined by binding to guanine nucleotides. In the GDP-bound state, Rab forms a cytoplasmic complex with Rab-GDI, 2 which regulates association with cellular components. After dissociation of Rab-GDI by a GDI dissociation factor on donor membranes, Rab replaces GDP with GTP through its interaction with a GEF at target membranes. This causes a conformational change in Rab that allows recruiting of a variety of downstream effectors onto membranes. During or after membrane fusion, a regulatory protein called Rab-GAP enhances the intrinsic GTPase activity of Rab and promotes GTP hydrolysis. Once this has occurred, GDP-bound Rab re-forms the complex with Rab-GDI and dissociates from membranes.Rab5, the most thoroughly characterized member of the Rab family, is localized mainly to early endosomes (4 -6). Rab5 is involved in the homotypic fusion process of early endosomes and in the budding of clathrin-coated vesicles from plasma membranes and transport to early endosomes (7-9). Therefore, Rab5-GEF plays a key role in the regulation of Rab5 function. To...

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Structural basis for Rab GTPase activation by VPS9 domain exchange factors

Cited by 124 publications

References 55 publications

Rabaptin-5-independent Membrane Targeting and Rab5 Activation by Rabex-5 in the Cell

Rabaptin-5-independent Membrane Targeting and Rab5 Activation by Rabex-5 in the Cell

The role of the hypervariable C-terminal domain in Rab GTPases membrane targeting

Characterization of RIN3 as a Guanine Nucleotide Exchange Factor for the Rab5 Subfamily GTPase Rab31

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