Kinetics of Interaction of Rab5 and Rab7 with Nucleotides and Magnesium Ions

Abstract

We describe here the kinetics of the interaction of GTP and GDP with the small GTP-binding proteins Rab5 and Rab7. It was possible to make use of the intrinsic fluorescence of these proteins, since Rab5 contains two and Rab7 three tryptophan residues, respectively. With both enzymes, there is a significant decrease in fluorescence on binding GTP and an increase on binding GDP. As with the small GTP-binding protein Ha-Ras p21 and with EF-Tu, nucleotide binding occurs in at least two steps and is describable in terms of a relatively weak initial interaction followed by a highly irreversible isomerization of the protein-nucleotide complex, which results in a change in the fluorescence properties. Dissociation of GDP and GTP could be followed in a time-dependent manner using fluorescently labeled GDP (methylanthraniloyl GDP) as displacing agent and taking advantage of substantial fluorescent energy transfer from tryptophan to the nucleotide. Fluorescence techniques could also be used to quantitate the interaction of Mg 2؉ ions with the GTP and GDP forms of Rab7, and it was shown that the metal ion was bound ϳ1000-fold more strongly to the GTP than the GDP form. The rate of GTP cleavage by the two proteins differed by a factor of ϳ20 (2 ؋ 10 ؊3 s ؊1 for Rab5 and 9 ؋ 10 ؊4 s ؊1 for Rab7 at 37°C). Both proteins showed significant discrimination against xanthosine 5-O-diphosphate (K d ϳ10 3 -fold higher than that of GDP) and dramatic discrimination against ADP or ATP (K d ϳ10 6 -fold higher than that of GDP). The results demonstrate a high degree of mechanistic similarity between the Rab proteins and other GTP-binding proteins, which have been examined in detail, including Ha-Ras p21, Ran, and EF-Tu.The Rab proteins are members of the Ras superfamily of small GTP-binding proteins. They are involved in protein trafficking in the cell and have been implicated in the mechanisms by which transport vesicles identify and fuse with their target compartment (1-4). Like the other members of the Ras superfamily, and in common with other classes of GTP-binding proteins, they cycle between the GTP-and GDP-bound forms and appear to share at least some of the general properties of the family, including interaction with nucleotide exchange factors (Refs. 5-8), in some cases (and perhaps in all) with GTPaseactivating proteins (GAPs 1 ; Refs. 9 and 10) and with effector molecules. A class of molecules which appear to be critical for the role of Rab proteins are GDP dissociation inhibitors, which bind Rabs that are prenylated (doubly geranylgeranylated near the C terminus) in a specific manner and deliver them to their respective membranes, as well as retrieving them from these locations (11). A related molecule, the Rab escort protein (REP), is necessary for prenylation of Rabs (catalyzed by a geranylgeranyltransferase) and is also involved in delivery to, and retrieval of Rabs from, membrane compartments (12). Other molecules that interact with Rab proteins have also been identified. These include rabphilin-3A, which is a putative targ...

The signal recognition particle receptor of Escherichia coli (FtsY) has a nucleotide exchange factor built into the GTPase domain

“…3b, the slope decreases at higher concentrations, and the dependence can be well described by a hyperbolic equation. This behavior is similar to that seen with other GTPases, including Ras (17), EF-Tu (18), and Rab5 or Rab7 (19), and has been interpreted as arising from a two-step binding mechanism as shown in Scheme 1:…”

“…Other GTPases that have been investigated at this level all show at least two-step binding of nucleotides. The association kinetics of GDP with FtsY initially appear very similar to those of Ras (17), EF-Tu (18), and Rab (19), with a weak but rapid initial binding step being followed by a relatively slow isomerization reaction. The rates of this step (k ϩ2 in Scheme 1) are approximately 20 s Ϫ1 (Ras), 400 s Ϫ1 (EF-Tu), and 80 s Ϫ1 (Rab7), compared with 119 s Ϫ1 for FtsY.…”

Rab11 Is Required for Trans-Golgi Network–to–Plasma Membrane Transport and a Preferential Target for GDP Dissociation Inhibitor

“…In this re-spect it is interesting to note that rab5, which has a high intrinsic GTPase activity and rapidly cycles between the GTP and GDP-bound form on membranes (Rybin et al, 1996), is relatively insensitive to release by GDI under our assay conditions (our unpublished results). In contrast, both rab7 and rab11 have significantly slower intrinsic GTPase activities (Urbé and Parton, 1995;Simon et al, 1996), yet both are sensitive to release from membranes by excess GDI (our unpublished results). Taken together, the data hint that the factors involved in stable membrane recruitment, including GDI displacement factor and guanine nucleotide exchange factor, may be the most critical in dictating the GDP-/GTP-bound ratio of an individual rab protein.…”

Specificity of Arl2/Arl3 signaling is mediated by a ternary Arl3‐effector‐GAP complex

“…We assume similar values for Arl3 because they do normally not differ very much from 10 5 to 10 6 M À1 s À1 for different Ras-superfamily proteins. Even weakly binding analogues of GDP or weak binding mutants of Ras have similar association rate constants, and even the very weak binding ADP associates to Rab5 with a rate constant of 4 * 10 4 M À1 s À1 [24,25]. The affinity of Arl3 for both Table 1 Kinetic rate constants and affinities of Arl proteins to nucleotides GDP and GTP is thus estimated to be 0.06 nM and 3 nM, respectively instead of 48 lM for GTP determined previously [15], possibly due to refolding of unstable nucleotide-free protein or dissociation of prebound GDP.…”