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Department of Cell Biology and Physiology, Washington University
School of Medicine, St. Louis, Missouri 63110
Nuclear pore complexes (NPCs) are large proteinaceous portals for
exchanging macromolecules between the nucleus and the cytoplasm. Revealing how this transport apparatus is assembled will be critical for understanding the nuclear transport mechanism. To address this
issue and to identify factors that regulate NPC formation and dynamics,
a novel fluorescence-based strategy was used. This approach is based on
the functional tagging of NPC proteins with the green fluorescent
protein (GFP), and the hypothesis that NPC assembly mutants will have
distinct GFP-NPC signals as compared with wild-type (wt) cells. By
fluorescence-activated cell sorting for cells with low GFP signal from
a population of mutagenized cells expressing GFP-Nup49p, three
complementation groups were identified: two correspond to mutant
nup120 and gle2 alleles that result in
clusters of NPCs. Interestingly, a third group was a novel
temperature-sensitive allele of nup57. The lowered
GFP-Nup49p incorporation in the nup57-E17 cells resulted
in a decreased fluorescence level, which was due in part to a sharply
diminished interaction between the carboxy-terminal truncated
nup57pE17 and wt Nup49p. Interestingly, the
nup57-E17 mutant also affected the incorporation of a
specific subset of other nucleoporins into the NPC. Decreased levels of
NPC-associated Nsp1p and Nup116p were observed. In contrast, the
localizations of Nic96p, Nup82p, Nup159p, Nup145p, and Pom152p were not
markedly diminished. Coincidentally, nuclear import capacity was
inhibited. Taken together, the identification of such mutants with
specific perturbations of NPC structure validates this
fluorescence-based strategy as a powerful approach for providing insight into the mechanism of NPC biogenesis.
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