IDENTIFICATION OF A YEAST KARYOPHERIN HETERODIMER THAT TARGETS IMPORTSUBSTRATE TO MAMMALIAN NUCLEAR-PORE COMPLEXES

Targeting of import substrate to nuclear pore complexes of permeabiliz ed vertebrate cells was previously shown to require a protein complex composed of two subunits, termed karyopherin. Yeast contain a homologu e of karyopherin alpha named Srp1p, which was initially identified as a genetic Suppressor of mutations in a subunit of RNA polymerase I. To determine whether yeast contain a karyopherin complex that includes S rp1p as the karyopherin alpha homologue, we genetically replaced Srp1p with a Srp1-Protein A chimera, Cytosol from this strain contained a c omplex, composed of the chimera and a protein of 95 kDa, that was puri fied using affinity chromatography on IgG Sepharose, Microsequence ana lysis showed that the 95-kDa protein was identical with a yeast protei n encoded by gene L8300.15 on chromosome XII. Sequence comparison reve aled that the L8300.15 gene product is the closest structural homologu e of vertebrate karyopherin beta. The yeast alpha and beta karyopherin subunits were expressed in Escherichia coli and were purified, When c ombined, they formed a heterodimeric complex and were active in target ing import substrate to nuclear envelopes of mammalian cells. We propo se that all karyopherins function as alpha/beta heterodimers.