The Kinetic Mechanism of Ran-Nucleotide Exchange Catalyzed by RCC1

Abstract

The interaction of Ran, a Ras-related nuclear GTP-binding protein, with its guanine nucleotide exchange factor RCC1 has been studied by equilibrium and transient kinetic measurements using fluorescent nucleotides. The four-step mechanism of catalyzed nucleotide exchange involves the formation of ternary complexes consisting of Ran, RCC1, and GXP as well as a nucleotide-free dimeric Ran.RCC1 complex. This model is sufficient to describe all experimental data obtained, so that no additional reaction steps must be assumed. All the rate and equilibrium constants for the four-step mechanism have been determined either experimentally or from a simultaneous theoretical fit to all experimental data sets. The affinities of RCC1 to Ran.GDP and Ran.GTP are similar (1.3 x 10(5) and 1.8 x 10(5) M-1, respectively) and are high enough to allow formation of the ternary complex under appropriate concentration conditions. In the absence of excess nucleotide and at low Ran concentrations, GDP (or GTP) can be efficiently displaced by excess RCC1 and the ternary complex can be produced. The affinities of both nucleotides (GDP or GTP) to Ran in the corresponding ternary complexes are reduced by orders of magnitude in comparison with the respective binary complexes. The reduction of affinity of both nucleotides in the ternary complexes leads to a dramatic increase in the dissociation rate constants by similar orders of magnitude (from 1.5 x 10(-5) s(-1) to 21 s(-1) for GDP) and thus to facilitated nucleotide exchange. The quantitative results of the kinetic analysis suggest that the exchange reaction does not per se favor the formation of the Ran.GTP complex, but rather accelerates the formation of the equilibrium dictated by the relative affinities of Ran for GDP/GTP and the respective concentrations of the nucleotide in the cell. The extent of Ran.GTP formation in vivo can be calculated using the constants derived.

Intermediates in the Guanine Nucleotide Exchange Reaction of Rab8 Protein Catalyzed by Guanine Nucleotide Exchange Factors Rabin8 and GRAB

“…Rabin8 has a similar catalytic efficiency as other GEFs, with GDP/GTP exchange accelerations in the range of ϳ10 2 -10 4 -fold, but some GEFs are considerably faster. Among these are Cdc25 (for Ras; acceleration factor of ϳ2 ϫ 10 5 ), DrrA from L. pneumophila (for Rab1b; ϳ8 ϫ 10 5 -fold), and RCC1 (for Ran; ϳ1.3 ϫ 10 6 -fold) (26,27,37). It has recently been pointed out that in many GTPase⅐GEF complexes of the Ras superfamily, the conserved lysine of the P-loop (Lys-21 in Rab8 and Lys-16 in H-Ras), which interacts with the ␤-phosphate of GDP or GTP, is intermediately stabilized by a conserved glutamate in the G3 motif at the beginning of switch II (DxxGQE motif; Glu-62 in Ras) (38).…”

Small GTP-binding protein Ran is regulated by posttranslational lysine acetylation

“…The fluorescence signal was plotted over time and fitted to a single-exponential function to give the observed rates (k obs ) for each RCC1 concentration. The plot of the k obs values over the RCC1 concentration resulted in a hyperbolic curve, which on fitting to a hyperbolic function resulted in the maximal rate of nucleotide dissociation, k -2 (29). The association of Ran•mantGppNHp and Importin-β was monitored by using an excitation of 350 nm and an emission cutoff filter of 420 nm; 200 nM (final 100 nM) of Ran•mantGppNHp was titrated with increasing concentrations of Importin-β (1-8 μM; final 0.5-4 μM).…”

“…The primary data were fitted to a single exponential function to result in the observed rate constants k obs . These k obs values were plotted against the RCC1 concentration following a hyperbolic function (29). The hyperbolic fit resulted in the rate of dissociation of the nucleotide from the ternary RCC1•Ran•mantGDP complex, k −2 ( Fig.…”

Reduction of exportin 6 activity leads to actin accumulation via failure of RanGTP restoration and NTF2 sequestration in the nuclei of senescent cells

“…Moreover, asymmetric distribution of Ran, RCC1, RanGAP1 and RanBP1 [46][47][48] results in a steep RanGTP gradient across the nuclear envelope; High RanGTP concentration in the nucleus, whereas high RanGDP concentration in the cytoplasm [30]. Furthermore, Ran is required directly for nuclear export rather than for re-import of potential export mediators [30].…”