Posttranslational modifications of Rab GTPases help their insertion into membranes.

G TPases of the Rab family, which comprises more than 60 members in mammals, have emerged as key regulators of virtually all transport steps between intracellular compartments. By interacting with a diverse range of effector proteins, such as lipid kinases and phosphatases, molecular motors, tethering factors, and scaffolding proteins, they regulate the formation of transport carriers from donor membranes, their movement along cytoskeletal tracks, and their targeting to acceptor membranes (1). Like other GTPases, Rab proteins function as molecular switches, cycling between an inactive GDP-bound form and an active GTP-bound form. Because Rabs display high affinities for both GDP and GTP and very low intrinsic GTPase activity, the GDP/GTP cycle must be regulated by guanine nucleotide exchange factors (GEFs) that promote GDP release and by GTPase-activating proteins (GAPs) that stimulates GTP hydrolysis. A salient feature of Rab proteins is a cycle of association/dissociation to/from membranes that is superimposed to the GDP/GTP cycle (Fig. 1A). Rabs therefore mainly exist as two pools, one membrane and GTP-bound and one cytosolic and GDP-bound (estimated depending on the Rab to range between 10% and 40% at the steady state). The stable insertion of Rabs in the outer leaflet of intracellular membranes is mediated by one or, in most cases, two geranylgeranyl lipid moieties that are attached to cysteine residue(s) present at their C terminus. This irreversible posttranslational modification occurring on newly synthesized Rabs involves a protein named REP (Rab escort protein), which presents the Rab proteins to Rab geranylgeranyl transferase (RabGGTase). Cytosolic Rabs are bound to the protein GDI (GDP Dissociation Inhibitor; two GDIs exist in mammals, GDIα/GDI-1 and GDIβ). GDI binds to Rabs with very high affinity (Kd in the nanomolar range) when they are prenylated and in the GDP state, which explains that GDI functions as a chaperone during the Rab cycle, extracting them from membranes after GAP-induced GTP hydrolysis (Fig. 1A, pathway 1) and solubilizing them in the form of RabGDP–GDI complexes. In a study in PNAS, Oesterlin et al. (2) address the question of Rab membrane attachment mechanism—the less well understood Rab cycle step. The Rab membrane attachment step couples, in fact, two processes: the targeting of different Rabs to specific membranes and their activation (GDP/GTP exchange), with both processes being intimately linked. Given the high affinity of GDI for prenylated RabGDP and consequently the low intrinsic rate of GDI dissociation, certainly the crucial event of this step is the dissociation of RabGDP– GDI complexes. Two main mechanisms have been proposed to explain GDI displacement. Based on the observations that nucleotide exchange occurs shortly after binding of purified Rab5 and Rab9– GDI complexes to endosomal membranes using in vitro or semi-in vitro assays (3, 4), the first mechanism suggests the existence of membrane-associated proteins termed GDF (GDI Displacement Factor) that favor the release of GDI (Fig. 1A, pathway 2). One protein with a GDF activity has been identified, Yip3/PRA1, and shown to achieve the dissociation of Rab9–GDI complexes and to facilitate Rab9 membrane insertion (5). However, it is difficult to generalize this mechanism, as Yip3 remains the only GDF identified so far. A second mechanism postulates that nucleotide exchange plays a central role in GDI release from RabGDP (Fig. 1B, pathway 3). It is based on the study of DrrA/SidM, an effector of Legionella pneumophila. Legionella effectors reorient the host cell vesicle-trafficking pathway to grab the host ER-derived vesicles necessary for the formation and maintenance of a specialized vacuolar compartment called Legionella-containing vacuole (LCV) (6, 7). DrrA/SidM specifically localizes, immediately after infection, to the LCV membrane, where it can efficiently recruit Rab1, a Rab regulating the early secretory pathway (8). Schoebel et al. first showed that the DrrA GEF domain is sufficient to displace GDI from Rab1GDP–GDI complex by catalyzing nucleotide exchange to GTP (9). Wu et al. then demonstrated, by using a semisynthetic farnesyl-NBD Rab7 probe, that the affinity of GDI for Rab7GTP is approximately three orders of magnitude lower than the affinity for Rab7GDP. Based on these results, the authors proposed that GEFs are necessary and sufficient for Rab membrane association (10). However, the GEFs cannot Fig. 1. (A) The Rab GTPase cycle. (B) Rab modifications by Legionella proteins dramatically change Rab– GDI complex affinity. A Rab protein prenylated with a fluorescently labeled farnesyl moiety (red star) forms a high-affinity complex with GDI (B, 1); the intrinsic dissociation rate of the complex is very low (B, 1 and 2), and equilibrium is shifted toward RabGDP–GDI complex (gray arrows). DrrA GEF activity converts Rab to the GTP-bound form (B, 2 and 3) snatching a small fraction of the RabGDP spontaneously released from GDI. The GTP loaded Rab has low affinity to GDI, and the equilibrium shifts toward complex dissociation. The activated Rab is recognized as a substrate by DrrA ATase domain that adenylylates the GTPase (B, 3 and 4). AnkX also relies on the free Rab pool generated by intrinsic Rab–GDI complex dissociation. The phosphocholine transferase modifies the Rabs (B, 2 and 5), rendering them incompetent to rebind to GDI and shifts the equilibrium to the complex dissociation state, whereas the lpg0696 dephosphocholination activity reverses the process (i.e., 5, then 2, in B).