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J. Cell Biol.,
Volume 143, Number 3, November 2, 1998 577-588
* M.E. Müller Institute for Microscopy, Biozentrum, University of Basel, CH-4056 Basel, Switzerland; and The nuclear pore complex (NPC), a supramolecular assembly of ~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 ~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.
University of
Heidelberg, Biochemie-Zentrum Heidelberg, D-69120 Heidelberg, Germany
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