MOLECULAR ARCHITECTURE OF THE YEAST NUCLEAR-PORE COMPLEX - LOCALIZATION OF NSP1P SUBCOMPLEXES

The nuclear pore complex (NPC), a supramolecular assembly of similar t o 100 different proteins (nucleoporins), mediates bidirectional transp ort of molecules between the cytoplasm and the cell nucleus. Extensive structural studies have revealed the three-dimensional (3D) architect ure of Xenopus NPCs, and eight of the similar to 12 cloned and charact erized vertebrate nucleoporins have been localized within the NPC. Tha nks to the power of yeast genetics, 30 yeast nucleoporins have recentl y been cloned and characterized at the molecular level. However, the l ocalization of these nucleoporins within the 3D structure of the NPC h as remain elusive, mainly due to limitations of preparing yeast cells for electron microscopy (EM). We have developed a new protocol for pre paring yeast cells for EM that yielded structurally well-preserved yea st NPCs. A direct comparison of yeast and Xenopus NPCs revealed that t he NPC structure is evolutionarily conserved, although yeast NPCs are 15% smaller in their linear dimensions. With this preparation protocol and yeast strains expressing nucleoporins tagged with protein A, we h ave localized Nsp1p and its interacting partners Nup49p, Nup57p, Nup82 p, and Nic96p by immuno-EM. Accordingly, Nsp1p resides in three distin ct subcomplexes which are located at the entry and exit of the central gated channel and at the terminal ring of the nuclear basket.