Molecular architecture of the yeast nuclear pore complex: localization of Nsp1p subcomplexes.
The nuclear pore complex (NPC), a supramolecular assembly of approximately 100 different proteins (nucleoporins), mediates bidirectional transport of molecules between the cytoplasm and the cell nucleus. Extensive structural studies have revealed the three- dimensional (3D) architecture of Xenopus NPCs, and eight of the approximately 12 cloned and characterized vertebrate nucleoporins have been localized within the NPC. Thanks to the power of yeast genetics, 30 yeast nucleoporins have recently been cloned and characterized at the molecular level. However, the localization of these nucleoporins within the 3D structure of the NPC has remain elusive, mainly due to limitations of preparing yeast cells for electron microscopy (EM). We have developed a new protocol for preparing yeast cells for EM that yielded structurally well-preserved yeast NPCs. A direct comparison of yeast and Xenopus NPCs revealed that the 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 have localized Nsp1p and its interacting partners Nup49p, Nup57p, Nup82p, and Nic96p by immuno-EM. Accordingly, Nsp1p resides in three distinct subcomplexes which are located at the entry and exit of the central gated channel and at the terminal ring of the nuclear basket.
Fahrenkrog
B
B
M.E. Müller Institute for Microscopy, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.
Hurt
E C
EC
Aebi
U
U
Panté
N
N
D000818:Animals; D002135:Calcium-Binding Proteins; D002467:Cell Nucleus / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D005656:Fungal Proteins / Q000032:analysis*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis; D009685:Nuclear Envelope / Q000737:chemistry; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000032:analysis*; D011993:Recombinant Fusion Proteins / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D014981:Xenopus
D002135:Calcium-Binding Proteins; D005656:Fungal Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; C082533:NIC96 protein, S cerevisiae; C062748:NSP1 protein, S cerevisiae; C077727:NUP49 protein, S cerevisiae; C091559:NUP57 protein, S cerevisiae; C095897:NUP82 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins