IDENTIFICATION OF A PROTEIN COMPLEX THAT IS REQUIRED FOR NUCLEAR-PROTEIN IMPORT AND MEDIATES DOCKING OF IMPORT SUBSTRATE TO DISTINCT NUCLEOPORINS

We have identified and characterized a 9S protein complex from a Xenop us ovary cytosolic subfraction (fraction A) that constitutes this frac tion's activity in recognizing a model nuclear import substrate and do cking it at the nuclear pore complex. Because of its function, the com plex is termed karyopherin. The 54- and 56-kDa subunits of the complex are termed alpha 1 and alpha 2, respectively, and the 97-kDa subunit is termed beta, In an alternative approach we have identified karyophe rin beta 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 beta, Compariso n with protein sequence data banks showed no significant similarity to other known proteins. Using nitrocellulose-immobilized rat liver nucl ear envelope proteins and nuclear import substrate as a ligand, we fou nd Xenopus fraction A-dependent binding to at least three bona fide nu cleoporins (Nup214, Nup153, and Nup98) and to a candidate nucleoporin with an estimated molecular mass of 270 kDa. We propose that these nuc leoporins function as docking proteins for karyopherin-mediated bindin g of substrate in a nuclear import/export pathway across the nuclear p ore complex.