RanBP1 Stabilizes the Interaction of Ran with p97 in Nuclear Protein Import

Three factors have been identified that reconstitute nuclear protein import in a permeabilized cell assay: the NLS receptor, p97, and Ran/TC4. Ran/ TC4, in turn, interacts with a number of proteins that are involved in the regulation of GTP hydrolysis or are components of the nuclear pore. Two Ran-binding proteins, RanBP1 and RanBP2, form discrete complexes with p97 as demonstrated by immunoadsorption from HeLa cell extracts fractionated by gel filtration chromatography. A >400-kD complex contains p97, Ran, and RanBP2. Another complex of 150-300 kD was comprised of p97, Ran, and RanBP1. This second trimeric complex could be reconstituted from recombinant proteins. In solution binding assays, Ran-GTP bound p97 with high affinity, but the binding of RanGDP to p97 was undetectable. The addition of RanBP1 with Ran-GDP or Ran-GTP increased the affinity of both forms of Ran for p97 to the same level. Binding of Ran-GTP to p97 dissociated p97 from immobilized NLS receptor while the Ran-GDP/RanBP1/p97 complex did not dissociate from the receptor. In a digitoninpermeabilized cell docking assay, RanBP1 stabilizes the receptor complex against temperature-dependent release from the pore. When added to an import assay with recombinant NLS receptor, p97 and Ran-GDP, RanBP1 significantly stimulates transport. These results suggest that RanBP1 promotes both the docking and translocation steps in nuclear protein import by stabilizing the interaction of Ran-GDP with p97. p ROTEINS targeted to the nucleus cross the nuclear envelope through a large proteinaceous supramolecular structure called the nuclear pore complex (NPC) 1 (Feldherr, 1984; reviewed by Newmeyer, 1993; Melchior and Gerace, 1995). The pore complex forms an aqueous channel between the nucleoplasm and cytoplasm allowing diffusion of small molecules <9 nm in diameter, but restricting the movement of larger macromolecules in both directions. Multiple transport mechanisms are thought to exist to transport diverse macromolecules from small proteins to large ribonucleoproteins (G6rlich and Mattaj, 1996). The import of nuclear proteins is mediated by single or bipartite stretches of primarily basic amino acid residues called nuclear localization sequences (NLSs) (Dingwall and Laskey, 1991). One of the earliest steps in nuclear protein import is recognition of the NLS by one of a family of cytoplasmic NLS-binding proteins of 54-56 kD known as the NLS receptor/importin edkaryopherin et (Adam and GerAddress all correspondence to S.A. Adam, Department of Cell and Molecular Biology, W129, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611. Tel." (312) 503-7525. Fax: (312) 5037912. 1. Abbreviat ions used in this paper: NLS, nuclear localization sequence; NPC, nuclear pore complex. ace, 1991; G6rlich et al., 1994; Weis et al., 1995). The NLScontaining proteins are then bound to the nuclear pore via a receptor complex containing the NLS receptor and a second factor, p97/importin [~/karyopherin [~ (Adam and Adam, 1994; G6rlich et al., 1995b; Moroianu et al., 1995a). It is thought that p97 mediates interaction with the pore by dimerization with the NLS receptor and direct association with a subset of a peptide repeat-containing family of nuclear pore complex proteins (nucleoporins) (Iovine et al., 1995; Moroianu et al., 1995b; Radu et al., 1995b). Interaction of the receptor with an NLS-containing protein is not required for translocation of the NLS receptor and p97 as a small region of the receptor that binds p97, the importin [3-binding domain (IBB), can direct nuclear accumulation of a chimeric protein (G6rlich et al., 1996; Weis et al., 1996). Subsequent release of the bound receptor complex and translocation through the nuclear pore complex requires the small GTPase Ran/TC4 and GTP hydrolysis (Melchior et al., 1993; Moore and Blobel, 1993). The translocation step in permeabilized cells is enhanced by the addition of another factor that interacts with Ran/TC4, pl0/ N'IT2 (Moore and Blobel, 1994; Paschal and Gerace, 1995). Ran/TC4, like other GTPases, is thought to act as a molecular switch (Bourne, 1990). The GTPase activity of Ran/TC4 is regulated by two proteins: the Ran nucleotide exchange factor, RCC1 (Bischoff and Ponstingl, 1991a,b), and the Ran GTPase-activating protein, RanGAP1 (Bis© The Rockefeller University Press, 0021-9525/96/11/559/11 $2.00 The Journal of Cell Biology, Volume 135, Number 3, November 1996 559-569 559 on N ovem er 9, 2017 jcb.rress.org D ow nladed fom choff et al., 1994; Becker et al., 1995). Mutational analysis of RCC1 and RanGAP1 has implicated both factors in protein import and RNA export (for review see Tartakoff and Schneiter, 1995; Corbett et al., 1995), although a direct linkage has not been shown and neither factor is required for import in permeabilized cells. A family of Ran-binding proteins containing conserved domains for interaction with Ran-GTP has been identified and includes RanBP1, RanBP2 (NUP358), Nup2p, and C. elegans F59A2.1 protein (Lounsbury et al., 1994; Beddow et al., 1995; Dingwall et al., 1995; Hartmann and Grrlich, 1995). Direct interactions between Ran-GTP and RanBP2 (Melchior et al., 1995a; Wu et al., 1995; Yokoyama et al., 1995), a XFXFG repeat-containing nucleoporin, and between Ran-GTP and RanBP1 (Coutavas et al., 1993; Lounsbury et al., 1994) have been demonstrated by protein overlay blot. Because of its peripheral cytoplasmic localization on the NPC and its ability to interact with RanGTP and p97, RanBP2 may be the initial docking site for nuclear protein import, thus committing the receptor complex for translocation across the nuclear pore complex (Melchior et al., 1995a). RanBP1 is involved in the coactivation of RanGTPase via an interaction with RanGAP1 and RanGTP (Ren et al., 1995; Bischoff et al., 1995; Richards et al., 1995) and inhibits GTP dissociation from Ran-GTP (Bischoff et al., 1995). Furthermore, RanBP1 can form a complex with nucleotide-free Ran/TC4 and the guanine nucleotide exchange factor, RCC1 (Bischoff et al., 1995; Hayashi et al., 1995; Saitoh and Dasso, 1995). Ran deletion mutants with the COOH-terminal DEDDDL sequence removed are unable to bind RanBP1 and are defective in RNA export, yet have no apparent effect on protein import (Ren et al., 1995; Richards et al., 1995). However, mutants in a yeast homologue of RanBP1, Yrblp, were found to be defective in both nuclear import and export (Schlenstedt et al., 1995b). We have investigated the role of the Ran-binding protein RanBP1 in nuclear protein import at the level of physical interactions with the nuclear transport factors p97 and Ran. Docking and transport experiments in digitonin permeabilized cells provide evidence that RanBP1 plays a role in the stabilization of a receptor complex during translocation through the pore by increasing the affinity of Ran-GDP for p97. Materials and Methods