We report the cDNA deduced primary structure of a wheat germ agglutinin-reactive nuclear pore complex (NPC) protein of rat. The protein, termed Nup98 (for nucleoporin of 98 kDa), contains numerous GLFG and FG repeats and some FXFG repeats and is thus a vertebrate member of a family of GLFG nucleoporins that were previously discovered in yeast. Immunoelectron microscopy showed Nup98 to be asymmetrically located at the nucleoplasmic side of the NPC. Nup98 functions as one of several docking site nucleoporins in a cytosolic docking activity-mediated binding of a model transport substrate. The docking site of Nup98 was mapped to its N-terminal half, which contains all of the peptide repeats. A recombinant segment of this region depleted the docking activity of cytosol. We suggest that the peptide repeat domain of Nup98, together with peptide repeat domains of other nucleoporins, forms an array of sites for mediated docking of transport substrate, and that bidirectional transport across the NPC proceeds by repeated docking and undocking reactions.

Although only 44% identical to human karyopherin a,, human karyopherin a2 (Rchl protein) substituted for human karyopherin a, ( (3, 4). Indeed, mapping of the docking site(s) for Nup98 showed it (them) to be located in the repeat-containing N-terminal half of Nup98 (12). Finally, the repeat-containing nucleoporins also contain binding sites for plO (M. Matunis, G.B., and M.H., unpublished data). It has been proposed that the repeatcontaining nucleoporins serve as a stationary phase and the transport factors as the mobile phase in transport across the NPC (12).We showed previously that the a subunit serves as an NLS receptor and proposed that the X3 subunit serves as an adaptor that binds to the a subunit-NLS substrate complex and to the repeat-containing nucleoporins (4). Here we show that the previously used karyopherin a subunit [corresponding to hSRP-1/NPI-1 (14, 15)], now termed karyopherin a1, can be replaced by karyopherin a2 [corresponding to the hSRP-1-related Rchl (16)]. Although karyopherin a, and a2 are only 44% identical, we did not detect any functional difference. We also show that karyopherin 3 is localized in the cytoplasm and at the nuclear rim and is largely lost after digitonin permeabilization of cells. Unlike karyopherin a, which alone cannot bind to the nuclear envelope of digitonin-permeabilized cells, karyopherin , can bind without karyopherin a being present. In an import reaction with all recombinant transport factors, the karyopherin a subunits, Ran, and plO are shown to enter the nucleus, whereas the ,B subunit remains at the nuclear envelope. This result suggests that the heterodimeric karyopherin complex is dissociated during transport. Overlay binding assays showed that the ,B subunit binds to the repeatcontaining nucleoporins. These data are consistent with the previously proposed function of karyopherin J3 as an adaptor between karyopherin a/NLS substrate complex and repeatcontaining nucleoporins.Abbreviations: HSA, human serum albumin; NLS, nuclear localization sequence; NPC, nuclear pore complex; FITC, fluorescein isothiocyanate.

We have identified and characterized a 9S protein complex from a Xenopus ovary cytosolic subfraction (fraction A) that constitutes this fraction's activity in recognizing a model nuclear import substrate and docking it at the nuclear pore complex. Because of its function, the complex is termed karyopherin. The 54-and 56-kDa subunits of the complex are termed al and a2, respectively, and the 97-kDa subunit is termed P. In an alternative approach we have identified karyopherin P from a rat liver cytosolic subfraction A by using immobilized rat nucleoporin Nup98 in a single, affinity-based enrichment step. We have molecularly cloned and sequenced rat karyopherin 13. Comparison with protein sequence data banks showed no significant similarity to other known proteins. Using nitrocellulose-immobilized rat liver nuclear envelope proteins and nuclear import substrate as a ligand, we found Xenopus fraction A-dependent binding to at least three bona fide nucleoporins (Nup214, Nupl53, and Nup98) and to a candidate nucleoporin with an estimated molecular mass of 270 kDa. We propose that these nucleoporins function as docking proteins for karyopherin-mediated binding of substrate in a nuclear import/export pathway across the nuclear pore complex.The majority of nuclear proteins contain a nuclear localization sequence (NLS) in their primary structure that is both necessary and sufficient for nuclear import (reviewed in refs. 1 and 2). In an in vitro import assay using digitonin-permeabilized cells, import-competent nuclei are retained but enough of the cytosolic proteins leak out so as to make import of an NLScontaining substrate dependent on exogenously added cytosol (3). This has allowed fractionation of the cytosol and identification of soluble proteins that complement the leaked-out cytosolic activities.Adam and Gerace (4) purified two proteins of 54 and 56 kDa from bovine erythrocyte cytosol on the basis of their ability to be cross-linked to an NLS-containing peptide. These two proteins, termed NLS receptors, will stimulate import activity of unfractionated cytosol but exhibit no detectable activity when added alone. Subsequently, Adam and Adam (5) purified a third bovine erythrocyte protein of 97 kDa (p97) that together with the NLS receptors stimulates binding of the import substrate at the nuclear envelope.Using a different approach, Moore and Blobel (6) showed that Xenopus ovary cytosol can be biochemically fractionated to yield two fractions (A and B) that are sufficient to satisfy the cytosolic requirement in digitonin-permeabilized mammalian cells and that exhibit distinct activities in the import assay. Fraction A is required for recognition of the NLS substrate and its docking at the nuclear envelope, while fraction B is required for translocation of the docked substrate into the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.nucleus. The two ...