The nuclear Ras-related protein Ran binds guanine nucleotide and is involved in cell cycle regulation. Models of the signal pathway predict Ran to be active as Ran GTP at the initiation of S phase upon activation by the nucleotide exchange factor RCC1 and to be inactivated for the onset of mitosis by hydrolysis of bound GTP. Here a nuclear homodimeric 65-kDa protein, RanGAPl, is described, which we believe to be the immediate antagonist of RCC1. It was purified from HeLa cell lysates and induces GTPase activity of Ran, but not Ras, by more than 3 orders of magnitude. The Ran mutant Q69L, modeled after RasQ61L, which is unable to hydrolyze bound GTP, is insensitive to RanGAP1.Ras-related small guanine nucleotide-binding proteins participate in various intracellular signal pathways. As a rule, the GTP-bound form represents the active state of a signal. Their latent GTPase activity is induced upon interaction with GTPase-activating proteins (GAPs). The GDP-bound form is inactive in signaling, and nucleotide exchange factors catalyze reactivation by exchanging GDP for GTP. Both nucleotides are otherwise firmly bound in the presence of Mg2+. Whereas most members of the Ras superfamily are associated with membranes, Ran (1) [or TC4, which was identified by homology screening in a human teratocarcinoma cDNA library (2)] is a soluble nuclear protein. In the GTP-bound form it is believed to be involved in inhibiting the onset of mitosis. Cells expressing mutated Ran/TC4 incapable of hydrolyzing GTP are arrested in G2 (3). In contrast, inactivation of its nucleotide exchange factor RCC1 results in premature chromosome condensation, activation of the cdc2 kinase, and onset of mitosis in S-phase cells (4), presumably by depletion of active Ran-GTP. Additional effects of inactivating or removing RCC1 have been described in various systems: Exit from mitosis is inhibited (5), G1 cells do not enter S phase (4), synthesis of double-stranded DNA is not initiated (6), and mRNA transcripts are processed incompletely (7-9). To identify further members of this signal pathway, we have used [y32P]GTP'Ran to monitor the isolation of a protein (RanGAP1) inducing its GTPase activity. MATERIALS AND METHODSPurification of RanGAP. Fifty milliliters of packed unsynchronized HeLa cells was thawed in 150 ml of lysis buffer [20 mM Bis-Tris-propane HCl, pH 7.0/1 mM EDTA/1 mM dithiothreitol (DTT)/protease inhibitors (10)] and swollen on ice for 20 min. They were lysed by 10 strokes with an S-type Dounce homogenizer and centrifuged at 70,000 x g for 60 min. The pellet was extracted with 100 mM NaCl in lysis buffer and recentrifuged at 70,000 x g for 30 min. Supernatants were pooled and chromatographed on Fractogel EMD DMAE-650/M (Merck; Superformance, 26 x 115 mm) in 20 mM Bis-Tris-propane HCl, pH 7.0/1 mM DTT with a linear gradient of NaCl from 0.05 M to 1 M at a flow rate of 5 ml/min. Fractions containing RanGAP were pooled and immediately applied to a hydroxylapatite column (Merck; Superformance, 10 x 150 mm) in 20 mM potassium phosphat...

The guanine nucleotide dissociation and GTPase reactions of Ran, a Ras-related nuclear protein, have been investigated using different fluorescence techniques to determine how these reactions are stimulated by the guanine nucleotide exchange factor RCC1 and the other regulatory protein, RanGAP1 (GTPase-activating protein). The intrinsic GTPase of Ran is one-tenth of the rate of p21ras and is even lower in the Ran(Q69L) mutant. Under saturating conditions the rate constant for the RanGAP1 stimulated GTPase reaction is 2.1 s-1 at 25 degrees C, which is a 10(5)-fold stimulation, whereas RanGAP1 has no effect on Ran(Q69L). The intrinsic guanine nucleotide dissociation rates of Ran are also very low and are likewise increased 10(5)-fold by the exchange factor RCC1. Methods to describe the reaction kinetically are presented. The Ran(T24N) mutant, which is analogous to the S17N mutant of p21ras, has decreased relative affinities for both GDP/GTP and favors GDP binding. However, it was found to interact almost normally with RCC1. The combination of these properties leads to stabilization of the Ran(T24N)-RCC1 complex and may result in vivo in depletion of RCC1 available for stimulating guanine nucleotide exchange.

The gene encoding the regulator of chromosome condensation (RCC1) was cloned by virtue of its ability to complement the temperature-sensitive phenotype of the hamster cell line tsBN2, which undergoes premature chromosome condensation or arrest in the G1 phase of the cell cycle at non-permissive temperatures. RCC1 homologues have been identified in many eukaryotes, including budding and fission yeast. Mutations in the gene affect pre-messenger RNA processing and transport, mating, initiation of mitosis and chromatin decondensation, suggesting that RCC1 is important in the control of nucleo-cytoplasmic transport and the cell cycle. Biochemically, RCC1 is a guanine-nucleotide-exchange factor for the nuclear Ras homologue Ran; it increases the dissociation of Ran-bound GDP by 10(5)-fold. It may also bind to DNAvia a protein-protein complex. Here we show that the structure of human RCC1, solved to 1.7-A resolution by X-ray crystallography, consists of a seven-bladed propeller formed from internal repeats of 51-68 residues per blade. The sequence and structure of the repeats differ from those of WD40-domain proteins, which also form seven-bladed propellers and include the beta-subunits of G proteins. The nature of the structure explains the consequences of a wide range of known mutations. The region of the protein that is involved in guanine-nucleotide exchange is located opposite the region that is thought to be involved in chromosome binding.

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.