Membrane Targeting of Rab GTPases Is Influenced by the Prenylation Motif

Gomes, Anita Quintal; Ali, Bassam R.; Ramalho, José S.; Godfrey, Richard F.; Barral, Duarte C.; Hume, Alistair N.; Seabra, Miguel C.

doi:10.1091/mbc.e02-10-0639

Cited by 136 publications

(124 citation statements)

References 65 publications

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“…S5C). These results suggest that digeranylgeranylation rather than the sequence of the prenylation motif is essential for the correct subcellular localization and function of Rab proteins, in keeping with a previous report on Rab5 membrane localization (27).…”

Section: Resultssupporting

confidence: 92%

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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Section: Resultssupporting

confidence: 92%

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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“…One is that the majority of Rabs are doubly geranylgeranylated and the second is that the enzyme exhibits highest affinity for mono-prenylated intermediates in the reaction (16), raising the possibility that these inhibitors sequester the cellular pools of the enzyme in non-productive complexes. Additionally, mono-geranylgeranylation of normally di-geranylgeranylated Rabs, such as Rab5, leads to mistargeting to the endoplasmic reticulum, and loss-of-function (17). Therefore, even if a proportion of inhibitor-induced mono-GG Rabs were able to interact with membranes, they would not be present at the correct cellular location.…”

Section: Discussionmentioning

confidence: 99%

“…Recombinant His 6 and glutatathione S-transferase-tagged Rab proteins were purified by nickel-Sepharose or glutathione-Sepharose affinity as described previously (16,17). All recombinant proteins were snap frozen in small aliquots and stored at Ϫ80°C until use.…”

Section: Methodsmentioning

confidence: 99%

Phosphonocarboxylates Inhibit the Second Geranylgeranyl Addition by Rab Geranylgeranyl Transferase

Baron

Tavaré

Figueiredo

et al. 2009

Journal of Biological Chemistry

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Rab geranylgeranyl transferase (RGGT) catalyzes the posttranslational geranylgeranyl (GG) modification of (usually) two C-terminal cysteines in Rab GTPases. Here we studied the mechanism of the Rab geranylgeranylation reaction by bisphosphonate analogs in which one phosphonate group is replaced by a carboxylate (phosphonocarboxylate, PC). The phosphonocarboxylates used were 3-PEHPC, which was previously reported, and 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid ((؉)-3-IPEHPC), a >25-fold more potent related compound as measured by both IC 50 and K i . (؉)-3-IPEHPC behaves as a mixed-type inhibitor with respect to GG pyrophosphate (GGPP) and an uncompetitive inhibitor with respect to Rab substrates. We propose that phosphonocarboxylates prevent only the second GG transfer onto Rabs based on the following evidence. First, geranylgeranylation of Rab proteins ending with a single cysteine motif such as CAAX, is not affected by the inhibitors, either in vitro or in vivo. Second, the addition of an -AAX sequence onto Rab-CC proteins protects the substrate from inhibition by the inhibitors. Third, we demonstrate directly that in the presence of (؉)-3-IPEHPC, Rab-CC and Rab-CXC proteins are modified by only a single GG addition. The presence of (؉)-3-IPEHPC resulted in a preference for the Rab N-terminal cysteine to be modified first, suggesting an order of cysteine geranylgeranylation in RGGT catalysis. Our results further suggest that the inhibitor binds to a site distinct from the GGPP-binding site on RGGT. We suggest that phosphonocarboxylate inhibitors bind to a GG-cysteine binding site adjacent to the active site, which is necessary to align the mono-GG-Rab for the second GG addition. These inhibitors may represent a novel therapeutic approach in Rab-mediated diseases.Most proteins of the Ras-like GTPase superfamily need to be post-translationally modified by prenyl groups to associate with cellular membranes and to activate downstream effectors (1). Protein prenylation involves the formation of a thioether link between conserved C-terminal cysteines in protein substrates and farnesyl pyrophosphate (FPP) 7 or geranylgeranyl pyrophosphate (GGPP) (2, 3). These prenyl pyrophosphates, which originate from the mevalonate pathway are utilized by three different prenyltransferase enzymes (2, 3). Farnesyl transferase (FT) and geranylgeranyl transferase type I (GGT-I) transfer FPP or GGPP, respectively, to a cysteine residue in the context of a C-terminal CAAX motif, where C is a cysteine, A is an aliphatic residue, and X is any amino acid. X contributes significantly to substrate specificity in FT and GGT-I (2). Rab geranylgeranyl transferase (RGGT) is distinct from FT and GGT-I in that it specifically recognizes Rab proteins, which vary in their C terminus containing CCXX, XCXC, XXCC, CCXXX, CAAX, and other motifs (3). RGGT exhibits exquisite specificity for Rabs due to the strict requirement of Rab escort protein (REP), which is a general Rab-GDP binding protein (4). RGGT is a heterodimeric enzyme ...

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“…Cell lysates were analysed by western blotting with the anti-ErbB4 antibody (Abcam) and anti-actin. mAb 1479 internalization COS-7 cells were grown on coverslips and transfected with pcDNA3.1ErbB4JM-aCYT-2, pcDNA3.1ErbB4JM-bCYT-2 or kinase-dead pcDNA3.1ErbB4JM-aCYT-2K751R and Rab5a-GFP (Gomes et al, 2003). The cells were treated for 5 min or 2 h with 1 mg/ml of mAb 1479.…”

Section: Immunohistochemistrymentioning

confidence: 99%

Suppression of breast cancer cell growth by a monoclonal antibody targeting cleavable ErbB4 isoforms

Hollmén

Määttä

Bald³

et al. 2009

Oncogene

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Membrane Targeting of Rab GTPases Is Influenced by the Prenylation Motif

Cited by 136 publications

References 65 publications

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

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

Phosphonocarboxylates Inhibit the Second Geranylgeranyl Addition by Rab Geranylgeranyl Transferase

Suppression of breast cancer cell growth by a monoclonal antibody targeting cleavable ErbB4 isoforms

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