Abstract
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.
Highlights
The nuclear pore complex (NPC), a supramolecular assembly of ف100 different proteins, mediates bidirectional transport of molecules between the cytoplasm and the cell nucleus
Yeast will be an excellent system for mapping Nups and studying the functional roles of nucleoporins, because 30 Nups have already been identified and molecularly characterized, and several mutant strains that affect both import of nuclear proteins and export of RNA have been created. These Nups have been localized to the nuclear envelope (NE) rim by immunofluorescence microscopy, none of them have been localized within the 3D NPC architecture. This is in part due to limitations in electron microscopy (EM) sample preparation of yeast cells, and to the high crossreactivity of anti-Nup antibodies caused by the presence of highly antigenic repetitive sequence motifs within the amino acid sequence of nucleoporins
Because yeast is an excellent system to combine biochemical and genetic manipulations with functional assays, and because most of the nucleoporins identified to date are from yeast NPCs, we have started to dissect the molecular architecture of the yeast NPC
Summary
The nuclear pore complex (NPC), a supramolecular assembly of ف100 different proteins (nucleoporins), mediates bidirectional transport of molecules between the cytoplasm and the cell nucleus. Whereas in vertebrates only 11 nucleoporins have been identified and molecularly characterized to date, in yeast the number of known Nups has reached 30 (for review see Panté and Aebi, 1996b; Doye and Hurt, 1997; Fabre and Hurt, 1997) This rapid progress in identifying and characterizing yeast Nups is due to (a) the development of a biochemical procedure to bulk isolate yeast NPCs (Rout and Blobel, 1993), (b) the use of genetic screens designed both to identify proteins interacting with a known Nup (i.e., synthetic lethal screens) and to isolate transport defected mutants (for review see Fabre and Hurt, 1997), and (c) the recent completion of the yeast genome project. The Journal of Cell Biology, Volume 143, Number 3, November 2, 1998 577–588 http://www.jcb.org expected that in the near future all yeast Nups will be identified
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