Previously identified protein of uncertain function is karyopherin alpha and together with karyopherin beta docks import substrate at nuclear pore complexes.

Abstract

Previously, we had purified a cytosolic protein complex, termed karyopherin, that functions in docking import substrate at the nuclear envelope in digitoninpermeabilized cells and also had molecularly cloned and sequenced its 97-kDa 13 subunit. We now report that the karyopherin a subunit is the previously identified protein NPI-1/SRP-1 of hitherto uncertain function. Using purified recombinant karyopherin a or .8 subunit, we showed that neither karyopherin a nor karyopherin 1 alone was sufficient for docking of import substrate at the nuclear envelope. Docking occurred only when both subunits were present. Moreover, docking of import substrate by the two recombinant karyopherin subunits was productive, as it led to nuclear internalization of the docked substrate in the presence of additional, previously characterized cytosolic factors. In a binding assay using immobilized karyopherin a and .8 subunits and import substrate as a ligand, we found that only karyopherin a bound ligand. We suggest that karyopherin 1 functions as an adaptor that binds both to karyopherin a and to any of a large number of docking sites that are represented by a repetitive peptide motif containing nucleoporins on both the cytoplasmic and nucleoplasmic side of the nuclear pore complex (NPC), bidirectionally ferrying a complex of karyopherin a-substrate across the NPC.In an in vitro nuclear import system that utilizes a nuclear localization sequence (NLS)-containing import substrate and digitonin-permeabilized cells (retaining intact importcompetent nuclei but being largely depleted of cytosolic proteins), import is dependent on exogenous cytosol (1, 2). The required cytosolic factors appear to be highly conserved because an amphibian (Xenopus ovary) cytosol can compensate for leaked out cytosol from digitonin-permeabilized mammalian cells (2). Biochemical subfractionation of the Xenopus ovary cytosol yielded two fractions (A and B) with distinct activities (2). Fraction A functions in the recognition of the NLS-containing substrate and its docking at the nuclear envelope, whereas fraction B is required for subsequent nuclear internalization of the docked substrate (2). The active components of both Xenopus cytosolic subfractions have been purified. The small GTP-binding protein designated "Ran" (3) for ras-related nuclear protein and a Ran-interactive protein, plO (4), represent fraction B's activity, whereas a 9-S complex of three proteins of 54, 56, and 97 kDa represents fraction A's activity (5). Because of its function in docking the import substrate prior to nuclear internalization, the 9-S complex has been named karyopherin, with the 54-and 56-kDa proteins termed its a subunit and the 97-kDa protein named its 13 subunit (5). Using bovine erythrocyte cytosol and based on crosslinking by an NLS-containing peptide, two proteins of 54 and 56 kDa were purified and termed the NLS receptor(s) (7). Subsequent purification of a third bovine erythrocyte protein of 97 kDa (p97) showed that both p97 and p54/56 were required for docking...

GLE2, a Saccharomyces cerevisiae homologue of the Schizosaccharomyces pombe export factor RAE1, is required for nuclear pore complex structure and function.

“…Srplp (also known as Kap6Op (Enenkel et al, 1995)) is the yeast homologue of the vertebrate NLS receptor/importin a/karyopherin a/hSRP1 (Gorlich et al, 1994;Loeb et al, 1995;Moroianu et al, 1995a;Weis et al, 1995). Srplp binds to NLS-bearing proteins in the cytoplasm and is required for docking of a NLS protein-Srplp-Kap95p trimeric complex to the NPC (reviewed in Gorlich and Mattaj, 1996).…”

A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex.

“…Abbreviations used in this paper: BRL, buffalo rat liver (cells); EST, expressed sequence tagged; NEM, N-ethylmaleimide; NPC, nuclear pore complex; PCLF, pore complex lamina formation. Chi et al, 1995;Enenkel et al, 1995;Imamoto et al, 1995;Moroianu et al, 1995a;Paschal and Gerace, 1995;Radu et al, 1995). Recognition of nuclear localization signal (NLS)-containing substrates in the cytoplasm is mediated by the c~ subunit of the karyopherin-cd13 heterodimer (Enenkel et al, 1995;Moroianu et al, 1995a;Weis et al, 1995), and docking of the trimeric complex to the NPC is mediated by the 13 subunit (Moroianu et al, 1995b;Radu et al, 1995).…”

“…Chi et al, 1995;Enenkel et al, 1995;Imamoto et al, 1995;Moroianu et al, 1995a;Paschal and Gerace, 1995;Radu et al, 1995). Recognition of nuclear localization signal (NLS)-containing substrates in the cytoplasm is mediated by the c~ subunit of the karyopherin-cd13 heterodimer (Enenkel et al, 1995;Moroianu et al, 1995a;Weis et al, 1995), and docking of the trimeric complex to the NPC is mediated by the 13 subunit (Moroianu et al, 1995b;Radu et al, 1995). Peptide repeat-containing nucleoporins (Rout and Wente, 1994), which bind karyopherin-13 in vitro and are components of the cytoplasmic and nucleoplasmic fibers as well as the peripheries of the central channel, have been proposed to form an array of substrate/receptor docking sites (Radu et al, 1995).…”

The Auto-inhibitory Function of Importin α Is Essentialin Vivo

“…This classical NLS is typified by a cluster of basic amino acids (monopartite) or two clusters of basic amino acids separated by a 10 -12 amino acid linker (bipartite) (11,12). A heterodimeric import receptor, composed of importins ␣ and ␤ (also known as karyopherin ␣ and ␤), mediates the nuclear import of proteins that contain a classical NLS (13)(14)(15). Over the last several years many studies have led to a detailed model for the individual steps in the classic nuclear transport cycle (5, 16): 1) importin ␣ binds to the NLS-cargo to form a trimeric import complex with importin ␤; 2) this NLS-cargo/importin ␣/importin ␤ complex is targeted to the NPC by importin ␤; 3) the complex then translocates into the nucleus where it encounters RanGTP; 4) upon binding RanGTP, importin ␤ dissociates from NLS-cargo/importin ␣; 5) NLS-cargo is released from importin ␣; and 6) once cargo is released, importin ␣ is recycled to the cytoplasm by its export receptor, Cse1p/CAS, in a trimeric complex with RanGTP.…”