Interactions between a nuclear transporter and a subset of nuclear pore complex proteins depend on Ran GTPase

Proteins to be transported into the nucleus are recognized by members of th e importin-karyopherin nuclear transport receptor family. After docking at the nuclear pore complex (NPC), the cargo-receptor complex moves through th e aqueous pore channel. Once cargo is released, the importin then moves bac k through the channel for new rounds of transport. Thus, importin and expor tin, another member of this family involved in export, are thought to conti nuously shuttle between the nuclear interior and the cytoplasm. In order to understand how nuclear transporters traverse the NPC, we constructed funct ional protein fusions between several members of the yeast importin family, including Pse1p, Sxm1p, Xpo1p, and Kap95p, and the green fluorescent prote in (GFP). Complexes containing nuclear transporters were isolated by using highly specific anti-GFP antibodies. Pse1-GFP was studied in the most detai l. Pse1-GFP is in a complex with importin-alpha and -beta (Srp1p and Kap95p in yeast cells) that is sensitive to the nucleotide-bound state of the Ran GTPase. In addition, Pse1p associates with the nucleoporins Nsp1p, Nup159p , and Nup116p, while Sxm1p, Xpo1p, and Kap95p show different patterns of in teraction with nucleoporins. Association of Pse1p with nucleoporins also de pends on the nucleotide-bound state of Ran; when Ran is in the GTP-bound st ate, the nucleoporin association is lost. A mutant form of Pse1p that does not bind Ran also fails to interact with nucleoporins. These data indicate that transport receptors such as Pse1p interact in a Ran-dependent manner w ith certain nucleoporins, These nucleoporins may represent major docking si tes for Pse1p as it moves in or out of the nucleus,ia the NPC.