Characterization of the ternary complex between Rab7, REP‐1 and Rab geranylgeranyl transferase

Abstract

Geranylgeranylation is a post-translational modification of Rab GTPases that enables them to associate reversibly with intracellular membranes. Geranylgeranylation of Rab proteins is critical for their activity in controlling intracellular membrane transport. According to the currently accepted model for their action, newly synthesized Rab proteins are recruited by Rab escort protein (REP) and are presented to the Rab geranylgeranyl transferase (RabGGTase) which covalentely modifies the Rab protein with two geranylgeranyl moieties. After prenylation, the Rab protein remains in complex with REP and is delivered to the target membrane by the latter. In this work, we show that RabGGTase can form a stable complex with Rab7±REP in the absence of its lipid substrate geranylgeranyl pyrophosphate. In order to characterize this interaction, we developed three fluorescence assays reporting on the interaction of RabGGTase with the Rab7±REP complex. For this interaction we determined a K d value of about 120 nm.Association of RabGGTase with the Rab7±REP complex occurs with a rate constant of < 10 8 m 21´s21 . We demonstrate that the state of the nucleotide bound to Rab7 does not influence the affinity of RabGGTase for the Rab7±REP-1 complex. Finally, we address the issue of substrate specificity of RabGGTase. Titration experiments demonstrate that, in contrast with farnesyl transferase, RabGGTase does not recognize a defined C-terminal sequence motif. Experiments using Rab7 mutants in which the last 16 amino acids were either mutated or truncated revealed that the distal part of the C-terminus makes only a limited contribution to the binding affinity between RabGGTase and the Rab7±REP-1 complex. This demonstrates the functional dissimilarity between RabGGTase and geranylgeranyl transferase I and farnesyl transferase, which interact specifically with the C-terminus of their substrates. Based on these experiments, we propose that RabGGTase recognizes the overall structure arising from the association of Rab and REP and then`scans' the flexible C-terminus to position the proximal cysteines into the active site.Keywords: geranylgeranylation; prenyltransferase; Rab7; Rab geranylgeranyl transferase; REP.Rab proteins are members of the Ras superfamily of small GTP-binding proteins. Many of them were shown to be implicated in control of intracellular vesicular traffic [1,2]. For their function Rab proteins require modification by geranylgeranyl isoprenoids. This allows them to associate reversibly with a membrane in a tightly controlled fashion [3,4]. Covalent attachment of geranylgeranyl groups to two C-terminal cysteines is catalyzed by Rab geranylgeranyl transferase (RabGGTase).Mammalian RabGGTase (GGTase II) is a heterodimer composed of tightly associated a and b subunits of 60 and 38 kDa, respectively [5,6], and belongs to the family of protein prenyl transferases together with farnesyl transferase and geranylgeranyl transferase I (GGTase I) [7]. Substrates of farnesyl transferase include members of the Ras family, lamin b and...

Interaction analysis of prenylated Rab GTPase with Rab escort protein and GDP dissociation inhibitor explains the need for both regulators

“…Bovine ␣-RabGDI was expressed in SF9 cells by using the baculoviral expression system as a fusion with an N-terminal 6His tag and the tobacco etch virus (TEV) protease cleavage site. Protein was purified to homogeneity by a combination of Ni-NTA chromatography, proteolytic removal of the His tag and gel filtration as described for Rab7 GTPase (31). Recombinant REP-1 was expressed and purified as described in ref.…”

“…Recombinant REP-1 was expressed and purified as described in ref. 31. The ␤-subunit of rat RabGGTase (␤GGT) was cloned into the pGATEV-mod vector where it is expressed as an N-terminal fusion with a 6His-GST assembly (32).…”

Membrane Targeting of a Rab GTPase That Fails to Associate with Rab Escort Protein (REP) or Guanine Nucleotide Dissociation Inhibitor (GDI)*

“…Prenylation of Rab proteins is catalyzed by geranylgeranyltransferase type II (GGTase II) (17,18). GGTase II will prenylate the target cysteine residues only if the nascent Rab substrate is first bound to a carrier protein termed Rab escort protein (REP) (19,20). REP initially binds to the Rab protein while it is in the GDP-bound state (21).…”

Phosphoisoprenoids Modulate Association of Rab Geranylgeranyltransferase with REP-1

“…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 Rab7 was performed as described (5).…”

“…Construction of dansyl-labeled Rab7 was performed as described (5). The GDP-bound form of Rab7C205SC207S mutant was generated as described (15). In vitro prenylation and purification of doubly prenylated dansylated Rab7⅐REP-1 complex was performed as described (16).…”