Npap60/Nup50 Is a Tri-Stable Switch that Stimulates Importin-α:β-Mediated Nuclear Protein Import

export by binding directly to the exportin, Crm1, forming a complex that has a higher affinity for RanGTP and cargo than has Crm1 alone (Englmeier et al., 2001; Lind-say et al., 2001). Crm1:RanBP3:RanGTP:cargo exports Summary from the nucleus to the cytoplasm and is disassembled by RanBP1 and the Ran GTPase activating protein, Ran-Many nuclear-targeted proteins are transported through the nuclear pore complex (NPC) by the im-GAP. To accomplish these functions, RanBP3 behaves as a bi-stable switch that changes its binding mode to portin-␣:␤ receptor. We now show that Npap60 (also called Nup50), a protein previously believed to be a Crm1 at different stages of the transport cycle. Yrb2p may play a similar role in yeast (Taura et al., 1998). structural component of the NPC, is a Ran binding protein and a cofactor for importin-␣:␤-mediated im-We noticed that two other proteins, Nup2p in budding yeast and the mammalian Npap60, possess a similar port. Npap60 is a tri-stable switch that alternates between binding modes. The C terminus binds im-arrangement of domains as RanBP3, despite the absence of overall sequence similarities. Nup2p was origi-portin-␤ through RanGTP. The N terminus binds the C terminus of importin-␣, while a central domain binds nally described as a nucleoporin and has been implicated in the export of importin-␣ from the nucleus (Booth importin-␤. Npap60:importin-␣:␤ binds cargo and can stimulate nuclear import. Endogenous Npap60 can et al., 1999; Hood et al., 2000). Npap60 (also termed Nup50) has also been described as a nucleoporin that shuttle and is accessible from the cytoplasmic side of the nuclear envelope. These results identify Npap60 is associated with the nucleoplasmic side of the NPC and is essential for mouse development (Smitherman et as a cofactor for importin-␣:␤ nuclear import and as a previously unidentified subunit of the importin al., 2000). Nonetheless, Nup2p and Npap60/Nup50 may not be orthologs, as their overall sequence similarity is complex. very low (14%). It has been reported that Npap60/Nup50 can bind Crm1, and that anti-Npap60/Nup50 antibodies Introduction specifically inhibit Crm1-mediated export (Guan et al., 2000). Eukaryotic cells are defined by the presence of a membrane bound nucleus that separates transcription from We describe here a different, and unexpected function for Npap60/Nup50 as a cofactor for nuclear protein im-translation. Macromolecular cargo moves between the cytoplasmic and nuclear compartment through nuclear port, rather than as a nucleoporin. For this reason, we prefer the name Npap60. In our hands, Npap60 binds …