期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 111, 期 24, 页码 E2453-E2461出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1406459111
关键词
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资金
- National Institutes of Health
- Sanford Research startup funds [RO1GM102203, RO1GM102486, RO1EB014869]
- French National Research Agency [ANR-12-BSV2-0008-01]
- Fondation ARC pourla Recherche sur le Cancer
- Institutional Development Awards from the National Institute of General Medical Sciences
- National Institutes of Health [P20GM103548, P20GM103620]
- Agence Nationale de la Recherche (ANR) [ANR-12-BSV2-0008] Funding Source: Agence Nationale de la Recherche (ANR)
Proximity-dependent biotin identification (BioID) is a method for identifying protein associations that occur in vivo. By fusing a promiscuous biotin ligase to a protein of interest expressed in living cells, BioID permits the labeling of proximate proteins during a defined labeling period. In this study we used BioID to study the human nuclear pore complex (NPC), one of the largest macromolecular assemblies in eukaryotes. Anchored within the nuclear envelope, NPCs mediate the nucleocytoplasmic trafficking of numerous cellular components. We applied BioID to constituents of the Nup107-160 complex and the Nup93 complex, two conserved NPC subcomplexes. A strikingly different set of NPC constituents was detected depending on the position of these BioID-fusion proteins within the NPC. By applying BioID to several constituents located throughout the extremely stable Nup107-160 subcomplex, we refined our understanding of this highly conserved subcomplex, in part by demonstrating a direct interaction of Nup43 with Nup85. Furthermore, by using the extremely stable Nup107-160 structure as a molecular ruler, we defined the practical labeling radius of BioID. These studies further our understanding of human NPC organization and demonstrate that BioID is a valuable tool for exploring the constituency and organization of large protein assemblies in living cells.
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