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 the importin-karyopherin nuclear transport receptor family. After docking at the nuclear pore complex (NPC), the cargo-receptor complex moves through the aqueous pore channel. Once cargo is released, the importin then moves back through the channel for new rounds of transport. Thus, importin and exportin, another member of this family involved in export, are thought to continuously shuttle between the nuclear interior and the cytoplasm. In order to understand how nuclear transporters traverse the NPC, we constructed functional protein fusions between several members of the yeast importin family, including Pse1p, Sxm1p, Xpo1p, and Kap95p, and the green fluorescent protein (GFP). Complexes containing nuclear transporters were isolated by using highly specific anti-GFP antibodies. Pse1-GFP was studied in the most detail. 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 interaction with nucleoporins. Association of Pse1p with nucleoporins also depends on the nucleotide-bound state of Ran; when Ran is in the GTP-bound state, 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 with certain nucleoporins. These nucleoporins may represent major docking sites for Pse1p as it moves in or out of the nucleus via the NPC.
Seedorf
M
M
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, and The Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.
Damelin
M
M
Kahana
J
J
Taura
T
T
Silver
P A
PA
D001483:Base Sequence; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D049452:Green Fluorescent Proteins; D028884:Karyopherins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins; D009154:Mutation; D009685:Nuclear Envelope / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D020931:ran GTP-Binding Protein
D002352:Carrier Proteins; D017931:DNA Primers; D005656:Fungal Proteins; D028884:Karyopherins; D008164:Luminescent Proteins; D026901:Membrane Transport Proteins; D009687:Nuclear Proteins; C483822:PSE1 protein, S cerevisiae; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D020558:GTP Phosphohydrolases; D020931:ran GTP-Binding Protein