Year-end Sale % OFF 
Abstract
Rab geranylgeranyltransferase (RabGGTase or GGTase-II) catalyzes the post-translational prenylation of Rab proteins. Rab proteins are recognized as substrates only when they are complexed to Rab Escort Protein (REP). The classical model of prenylation complex assembly assumes initial formation of the Rab.REP binary complex, which subsequently binds to RabGGTase loaded with the isoprenoid donor geranylgeranyl pyrophosphate (GGpp). We demonstrate here that REP-1 can also associate with RabGGTase in the absence of Rab protein and that this interaction is dramatically strengthened by the presence of phosphoisoprenoids such as GGpp. The GGpp-dependent interaction between RabGGTase and REP-1 was observed using affinity precipitations and gel filtration and was quantitated on the basis of fluorescence assays. In the presence of GGpp, REP-1 binds to RabGGTase with a K(d) value of approximately 10 nm, while in its absence the affinity between the two proteins is in the micromolar range. We further demonstrate that binding of Rab7 to the RabGGTase.GGpp.REP-1 complex occurs without prior dissociation of REP-1. Analysis of binding and prenylation rate constants indicate that the RabGGTase.GGpp.REP-1 complex can function as a kinetically competent intermediate of the prenylation reaction. We conclude that, depending on the prevailing concentrations, binding of REP-1 to RabGGTase in the presence of GGpp may serve as an alternative pathway for the assembly of the prenylation machinery in vivo. Implications of these findings for the role of REP-1 in the prenylation reaction are discussed.
Highlights
Rab proteins are members of the Ras superfamily of small GTP-binding proteins, which are directly implicated in the regulation of vesicular traffic [1]
We demonstrate here that Rab Escort Protein (REP)-1 can associate with RabGGTase in the absence of Rab protein and that this interaction is dramatically strengthened by the presence of phosphoisoprenoids such as geranylgeranyl pyrophosphate (GGpp)
We further demonstrate that binding of Rab7 to the RabGGTase1⁄7 GGpp1⁄7REP-1 complex occurs without prior dissociation of REP-1
Summary
Protein Expression, Purification, and Modification—Expression of rat RabGGTase and REP-1 in SF21 cells and subsequent purification was performed as described [14, 15]. Construction of dansyl-labeled Rab was performed as described [5]. In vitro prenylation and purification of doubly prenylated dansylated Rab71⁄7REP-1 complex was performed as described [16]. Loading of Rab with mantGDP was performed as described [17]. Analytical Gel Filtration Chromatography—Protein complex formation was performed in 500 l of 40 mM Hepes, pH7.2, 150 mM NaCL, 5 mM DTE, 3 mM MgCL2, 90 M GGpp. The sample was mixed, centrifuged in a bench top centrifuge for 5 min, loaded onto a Superdex 200 10/20 gel filtration column driven by a Waters 490E HPLC system, and equilibrated with 40 mM Hepes, pH7.2, 150 mM NaCL, 5 mM DTE, 3 mM.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
