It has been shown before by (31)P NMR that Ras bound to the nonhydrolyzable GTP analogue guanosine 5'-O-(beta, gamma-imidotriphosphate) (GppNHp) exists in two conformations which are rapidly interconverting with a rate constant of 3200 s-1 at 30 degrees C [Geyer, M., et al. (1996) Biochemistry 35, 10308-10320]. Here we show that Ran complexed with GTP also exists in two conformational states, 1 and 2, which can be directly inferred from the occurrence of two (31)P NMR resonance lines for the gamma-phosphate group of bound GTP. The exchange between the two states is slow on the NMR time scale with a value of <200 s-1 at 5 degrees C for the corresponding first-order rate constants. In wild-type Ran, the equilibrium constant K' between the two states is 0.7 at 278 K, is different for various mutants, and is strongly dependent on the temperature. The standard enthalpy DeltaH degrees and the standard entropy DeltaS degrees for the conformational transitions determined from the NMR spectra are as follows: DeltaH degrees = 37 kJ mol-1 and DeltaS degrees = 130 J mol-1 K-1 for wild-type Ran.GTP. In complex with the Ran-binding protein RanBP1, one of the Ran.GTP conformations (state 2) is stabilized. The interaction of Ran with the guanine nucleotide exchange factor protein RCC1 was also studied by (31)P NMR spectroscopy. In the presence of nucleotide, the ternary complex of Ran.nucleotide.RCC1, an intermediate in the guanine nucleotide exchange reaction, could be observed. A model for the conformational transition of Ran.GTP is proposed where the two states observed are caused by the structural flexibility of the effector loop of Ran; in solution, state 2 resembles the GTP-bound form found in the crystal structure of the Ran-RanBP complex.

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Receptor-mediated nucleocytoplasmic transport is dependent on the GTPase Ran and Ran-binding protein 1 (RanBP1). The acidic C terminus of Ran is required for high affinity interaction between Ran and RanBP1. We found that a novel Ran mutant with four of its five acidic C-terminal amino acids modified to alanine (RanC4A) has an ϳ20-fold reduced affinity for RanBP1. We investigated the effects of RanC4A on nuclear import and export in permeabilized HeLa cells. Although RanC4A promotes accumulation of the nuclear export receptor CRM1 at the cytoplasmic nucleoporin Nup214, it strongly stimulates nuclear export of GFP-NFAT. Since RanC4A exhibits an elevated affinity for CRM1 and other nuclear transport receptors, this suggests that formation of the export complex containing CRM1, Ran-GTP, and substrate is a rate-limiting step in export, not release from Nup214. Conversely, importin ␣/␤-dependent nuclear import of bovine serum albumin, coupled to a classical nuclear localization sequence is strongly inhibited by RanC4A. Inhibition can be reversed by additional importin ␣, which promotes the formation of an importin ␣/␤ complex. These results provide physiological evidence that release of Ran-GTP from importin ␤ by RanBP1 and importin ␣ is critical for the recycling of importin ␤ to a transport-competent state.Molecular transport between the cytoplasm and the nucleus occurs through nuclear pore complexes (NPCs), 1 large supramolecular structures that span the nuclear envelope (for review, see Refs. 1 and 2). Small molecules such as ions and metabolites cross the NPC by passive diffusion. In contrast, most macromolecules are transported through the NPC by signal-and energy-dependent processes. Much of the signal-dependent nucleocytoplasmic transport is mediated by nucleocytoplasmic shuttling receptor proteins of the importin ␤/karyopherin ␤ family. These receptors are thought to transfer their cargoes between the nucleus and the cytoplasm by sequentially interacting with a series of NPC proteins (nucleoporins; for review, see Refs. 3 and 4).Transport mediated by importin/karyopherin ␤-type transport receptors is dependent on the small GTPase Ran, which shuttles between the cytoplasm and the nucleus (3). Due to the segregation of the RanGEF in the nucleus and the RanGAP in the cytoplasm, the GTP-bound form of Ran is thought to be concentrated in the nucleus and the GDP-bound form in the cytoplasm. Ran-GTP, which binds to all importin ␤-type import receptors, promotes the association of cargoes with export receptors and the dissociation of cargoes from import receptors in vitro and thus appears to regulate receptor loading and unloading in the nucleus (see below).The best characterized nuclear import pathway involves cargo proteins carrying a basic amino acid-rich nuclear localization sequence (NLS). A trimeric import complex is formed by the binding of the cargo to the adapter protein importin ␣, which interacts with the import receptor importin ␤ via its importin ␤-binding (IBB) domain (3). After the import complex is t...

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