Facilitated Nucleocytoplasmic Shuttling of the Ran Binding Protein RanBP1

The defining feature of eukaryotic cells is the compartmentalization of DNA replication and transcription within the nucleus. Access to the nuclear compartment is provided by pores that are plugged through the double membrane of the nuclear envelope (for reviews, see references 15 and 66). A multitude of soluble transport receptors and accessory proteins controls the transit of protein and nucleic acid cargo through these pores (for recent reviews, see references 24, 40, 42, and 64). The vectoriality of cargo transport is in many cases dependent on the asymmetric distribution of Ran, a small GTP binding protein, and of its regulatory factors (8, 17). Ran is predominantly nuclear and is believed to be maintained in the GTP-bound state (Ran:GTP) by a guanine nucleotide exchange factor, RCC1, which is associated with chromatin in the nucleus (7, 58). A Ran-specific GTPase-activating protein, RanGAP, which converts Ran to the GDP-bound state, is excluded from the nucleus (5, 13, 30). This asymmetric arrangement of regulatory factors ensures that a steep Ran:GTP gradient exists across the nuclear pores, and the collapse of this gradient inhibits many forms of nuclear traffic (32, 52). Proteins destined for import into the nucleus frequently possess a nuclear localization signal (NLS) containing multiple basic amino acid residues (16). The NLS is recognized by an adapter protein called importin α (also variously called karyopherin-α, p56, SRP1, PTAC58, and pendulin) which associates with a transporter module, importin β (also called karyopherin-β and p97) (11, 25, 31, 36, 48, 61). This complex can bind to nuclear pores and translocate from one side of the pore to the other, probably by a type of facilitated diffusion (18, 50). Ran:GTP, present within the nucleus, dissociates the complex from the pores and releases importin α and its associated cargo from importin β (26, 49). Thus, it may be that the Ran gradient is not required for translocation per se but can drive a net accumulation of the cargo in the nuclear compartment. The importin β is then believed to return to the cytosol as a complex with Ran:GTP (26, 28). There, the RanGAP must hydrolyze the GTP to permit release of the importin β for another round of import. However, RanGAP alone is unable to act on Ran:GTP that is bound to importin β (19, 26). Another cytosolic factor, the Ran binding protein RanBP1, is required together with importin α to permit efficient GTP hydrolysis by RanGAP (4, 20). The giant nucleoporin Nup358 can probably serve a similar function (65, 67). RanBP1 is a 23-kDa cytosolic protein that possesses a conserved Ran binding domain (RanBD) of about 135 amino acid residues, which specifically recognizes Ran in the GTP-bound state (3, 14). The crystal structure of Ran in a complex with the second RanBD of Nup358 has been solved, and the two proteins form an unusually tight embrace in which the N terminus of the RanBD is wrapped around Ran, and the C terminus of Ran is wrapped around the RanBD (62). Ran:GTP can form a ternary complex with RanBP1 and importin β or with other members of the karyopherin family, and this complex is only weakly susceptible to RanGAP (4, 38). The presence of importin α, which will sequester free importin β, increases the efficiency of the hydrolysis (4, 20). RanBP1 is therefore predicted to be required for continued nuclear protein import, and this has been shown to be the case in yeast (57). RanBP1 and importin β have no measurable affinity for each other or—separately—for Ran:GDP, but surprisingly they can form a stable ternary complex with one another (12). The purpose of this RanBP1-importin β-Ran:GDP complex remains obscure. Proteins that need to be exported from the nucleus often possess a distinct, leucine-rich nuclear export signal (NES) (for a review, see reference 23). The NES can form a ternary complex with an export receptor, called Crm1 (or exportin 1), and Ran:GTP (2, 21, 22, 44, 60). The formation of this complex will therefore be favored in the nucleus. After translocation through the pores, RanGAP—again with the likely assistance of RanBP1—can hydrolyze the Ran:GTP and release the NES cargo into the cytosol. Therefore, RanBP1 probably plays an essential role in nuclear protein export. A similar mechanism can account for the export of importin α by the transport factor Cas (37). A complete understanding of nuclear protein transport requires that we determine the mechanism by which essential factors such as RanBP1 maintain their specific locations within the cell. The role of RanBP1 as an accessory factor for RanGAP implies that RanBP1 must be retained in the cytosol. Indeed, ectopic expression of nuclear RanBP1 is toxic to cells, and microinjection of RanBP1 into nuclei inhibits nuclear transport, probably because it sequesters nuclear Ran:GTP and effectively collapses the nucleocytoplasmic Ran:GTP gradient (32, 53). It may also interfere with the formation of export complexes. This requirement for a cytoplasmic location presents a problem to the cell, because RanBP1 is in principle small enough to diffuse through the nuclear pores, and within the nucleus it will form a stable ternary complex with Ran:GTP and importin β. Two distinct mechanisms may confer the cytoplasmic distribution of small proteins: (i) retention by binding to a larger cytoplasmic protein and (ii) active export. RanBP1 possesses a functional NES near its C terminus (54, 68). However, it also possesses an adjacent upstream sequence that may act in cytoplasmic retention (54). Three other groups have provided indications that RanBP1 may shuttle between the cytosol and nucleus. Zolotukhin and Felber (68) found that the ectopic expression of the Rev protein (which contains an NES) can lead to the nuclear accumulation of RanBP1, presumably by competing for Crm1. Schlenstedt et al. (56), in a study of the Yrb4p importin in yeast, demonstrated that a dominant interfering mutant of Yrb4p that blocks bidirectional nuclear transport also caused the nuclear accumulation of Yrb1p, the yeast homologue of RanBP1. However, in both cases the inhibitory protein was expressed for a period greater than the cell cycle time. Thus, these results do not resolve whether RanBP1 continually shuttles across the nuclear pores or instead enters the nucleus only at a specific time during cell division. For example, in mammalian cells an NES might be required to ensure that RanBP1 is excluded from the nucleus after mitosis when the integrity of the nuclear envelope is reestablished. A third study examined the distribution of RanBP1 in Xenopus oocytes after microinjection of an NES-peptide conjugate, which inhibits mRNA and snRNA transport (46). Oocytes were fractionated after 6.5 h, and the nuclear and cytosolic fractions were immunoblotted for RanBP1. These results demonstrated that RanBP1 became partially associated with the nuclei—either at the pores or within the nucleoplasm—in the presence of the competing NES-peptide. In all of these studies, however, it could be argued that the RanBP1 enters the nucleus only in response to a general perturbation in nuclear transport. We now show that RanBP1 does continually shuttle quite rapidly between the nuclear and cytoplasmic compartments. Leptomycin B (LMB), which specifically inhibits NES-mediated export (63), causes a nuclear accumulation of endogenous RanBP1 and of a green fluorescent protein (GFP)-RanBP1 fusion protein. Microinjected GST-RanBP1 lacking its C-terminal NES accumulates in the nucleus in the absence of any transport inhibitors. Collapse of the Ran:GTP gradient results in the equilibration of RanBP1 across the nuclear envelope. The nuclear accumulation of a GST-RanBP1 mutant that lacks the NES requires nuclear Ran:GTP. A point mutation in the N-terminal arm of the RanBD of RanBP1 completely blocks import into nuclei under the conditions tested. Although this mutant has a reduced affinity for Ran, it can form a ternary complex with Ran:GTP and importin β or importin 5 (47). We propose that the RanBP1 can translocate through the nuclear pores by an active mechanism and, in the absence of functional export, accumulate within the nuclei in a complex with Ran:GTP.