Proteomic analysis of nucleoporin interacting proteins

The Saccharomyces cerevisiae nuclear pore complex is a supramolecular assem bly of 30 nucleoporins that cooperatively facilitate nucleocytoplasmic tran sport. Thirteen nucleoporins that contain FG peptide repeats (FG Nups) are proposed to function as stepping stones in karyopherin-mediated transport p athways. Here, protein interactions that occur at individual FG Nups were s ampled using immobilized nucleoporins and yeast extracts. We find that many proteins bind to FG Nups in highly reproducible patterns. Among 135 protei ns identified by mass spectrometry, most were karyopherins and nucleoporins . The PSFG nucleoporin Nup42p and the GLFG nucleoporins Nup49p, Nup57p, Nup 100p, and Nup116p exhibited generic interactions with karyopherins; each bo und 6-10 different karyopherin betas, including importins as well as export ins. Unexpectedly, the same Nups also captured the hexameric Nup84p complex and Nup2p. In contrast, the FXFG nucleoporins Nup1p, Nup2p, and Nup60p wer e more selective and captured mostly the Kap95p . Kap60p heterodimer. When the concentration of Gsp1p-GTP was elevated in the extracts to mimic the nu cleoplasmic environment, the patterns of interacting proteins changed; expo rtins exhibited enhanced binding to FG Nups, and importins exhibited reduce d binding. The results demonstrate a global role for Gsp1p-GTP on karyopher in-nucleoporin interactions and provide a rudimentary map of the routes tha t karyopherins take as they cross the nuclear pore complex.