title: Systematic Protein-Protein Interaction Analysis Reveals Intersubcomplex Contacts in the Nuclear Pore Complex.
abstract: The nuclear pore complex (NPC) enables transport across the nuclear envelope. It is one of the largest multiprotein assemblies in the cell, built from about 30 proteins called nucleoporins (Nups), organized into distinct subcomplexes. Structure determination of the NPC is a major research goal. The assembled ∼40-112 MDa NPC can be visualized by cryoelectron tomography (cryo-ET), while Nup subcomplexes are studied crystallographically. Docking the crystal structures into the cryo-ET maps is difficult because of limited resolution. Further, intersubcomplex contacts are not well characterized. Here, we systematically investigated direct interactions between Nups. In a comprehensive, structure-based, yeast two-hybrid interaction matrix screen, we mapped protein-protein interactions in yeast and human. Benchmarking against crystallographic and coaffinity purification data from the literature demonstrated the high coverage and accuracy of the data set. Novel intersubcomplex interactions were validated biophysically in microscale thermophoresis experiments and in intact cells through protein fragment complementation. These intersubcomplex interaction data provide direct experimental evidence toward possible structural arrangements of architectural elements within the assembled NPC, or they may point to assembly intermediates. Our data favors an assembly model in which major architectural elements of the NPC, notably the Y-complex, exist in different structural contexts within the scaffold.
authors.affiliation: From the ‡Otto-Warburg Laboratory, Max-Planck Institute for Molecular Genetics (MPIMG), Berlin, Germany;
authors.forename: Luise
authors.initials: L
authors.lastname: Apelt
authors.affiliation: §Department of Biology, Massachusetts Institute of Technology (MIT), Cambridge;
authors.forename: Kevin E
authors.initials: KE
authors.lastname: Knockenhauer
authors.affiliation: §Department of Biology, Massachusetts Institute of Technology (MIT), Cambridge;
authors.forename: Nina C
authors.initials: NC
authors.lastname: Leksa
authors.affiliation: From the ‡Otto-Warburg Laboratory, Max-Planck Institute for Molecular Genetics (MPIMG), Berlin, Germany;
authors.forename: Nouhad
authors.initials: N
authors.lastname: Benlasfer
authors.affiliation: §Department of Biology, Massachusetts Institute of Technology (MIT), Cambridge;
authors.forename: Thomas U
authors.initials: TU
authors.lastname: Schwartz
authors.affiliation: From the ‡Otto-Warburg Laboratory, Max-Planck Institute for Molecular Genetics (MPIMG), Berlin, Germany; ¶Institute of Pharmaceutical Sciences, Pharmaceutical Chemistry, University of Graz, Graz, Austria stelzl@molgen.mpg.de.
authors.forename: Ulrich
authors.identifier: 0000-0003-2500-3585
authors.initials: U
authors.lastname: Stelzl
mesh_terms: D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D028861:Nuclear Pore Complex Proteins / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D025941:Protein Interaction Mapping / Q000379:methods*; D055503:Protein Multimerization; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D028861:Nuclear Pore Complex Proteins; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins