MAMMALIAN KARYOPHERIN ALPHA(1)BETA AND ALPHA(2)BETA HETERODIMERS - ALPHA(1) OR ALPHA(2) SUBUNIT BINDS NUCLEAR-LOCALIZATION SIGNAL AND BETA-SUBUNIT INTERACTS WITH PEPTIDE REPEAT-CONTAINING NUCLEOPORINS

Although only 44% identical to human karyopherin alpha(1), human karyopherin alpha(2) (Rch1 protein) substituted for human karyopherin alpha(1) (hSRP-1/NPI-1) in recognizing a standard nuclear localization sequence and karyopherin beta-dependent targeting to the nuclear envelope of digitonin-permeabilized cells. By immunofluorescence microscopy of methanol-fixed cells, karyopherin beta was localized to the cytoplasm and the nuclear envelope and was absent from the nuclear interior. Digitonin permeabilization of buffalo rat liver cells depleted their endogenous karyopherin beta. Recombinant karyopherin beta can bind directly to the nuclear envelope of digitonin-permeabilized cells at 0 degrees C (docking reaction). In contrast, recombinant karyopherin alpha(1) or alpha(2) did not bind unless karyopherin beta was present. Likewise, in an import reaction (at 20 degrees C) with all recombinant transport factors (karyopherin alpha(1) or alpha(2), karyopherin beta, Ran, and p10) import depended on karyopherin beta. Localization of the exogenously added transport factors after a 30-min import reaction showed karyopherin beta at the nuclear envelope and karyopherin alpha(1) or alpha(2), Ran, and p10 in the nuclear interior. In an overlay assay with SDS/PAGE-resolved and nitrocellulose-transferred proteins of the nuclear envelope, S-35-labeled karyopherin beta bound to at least four peptide repeat-containing nucleoporins-Nup358, Nup214, Nup153, and Nup98.