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Abstract
The nuclear pore complex (NPC) is an enormous proteinaceous complex composed of multiple copies of about 30 different proteins called nucleoporins. In this study, we analyzed the composition of the NPC in the model organism Schizosaccharomyces pombe using strains in which individual nucleoporins were tagged with GFP. We identified 31 proteins as nucleoporins by their localization to the nuclear periphery. Gene disruption analysis in previous studies coupled with gene disruption analysis in the present study indicates that 15 of these nucleoporins are essential for vegetative cell growth and the other 16 nucleoporins are non-essential. Among the 16 non-essential nucleoporins, 11 are required for normal progression through meiosis and their disruption caused abnormal spore formation or poor spore viability. Based on fluorescence measurements of GFP-fused nucleoporins, we estimated the composition of the NPC in S. pombe and found that the organization of the S. pombe NPC is largely similar to that of other organisms; a single NPC was estimated as being 45.8–47.8 MDa in size. We also used fluorescence measurements of single NPCs and quantitative western blotting to analyze the composition of the Nup107-Nup160 subcomplex, which plays an indispensable role in NPC organization and function. Our analysis revealed low amounts of Nup107 and Nup131 and high amounts of Nup132 in the Nup107-Nup160 subcomplex, suggesting that the composition of this complex in S. pombe may differ from that in S. cerevisiae and humans. Comparative analysis of NPCs in various organisms will lead to a comprehensive understanding of the functional architecture of the NPC.
Disclosure of Potential Conflicts of Interest
No potential conflict of interest was disclosed.
Acknowledgments
We thank Shelly Sazer and Paul Nurse for mph1 and cds1 strains and Aki Hayashi, Chie Mori, Miho Yamane, and Kasumi Okamasa for their technical assistances. We also thank DB Alexander for critical reading of the manuscript. This work was supported by grants from the Japan Science and Technology Agency (to T.H.) and the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to H.A., Y.C., M.T., M.I., Y.H., and T.H.), and by a grant (H.A., Y.C., and M.I.) and a fellowship (H.-J. Y.) from the Japan Society for the Promotion of Science.