title: The stoichiometry of the nucleoporin 62 subcomplex of the nuclear pore in solution.
abstract: The nuclear pore complex (NPC) regulates transport between the nucleus and cytoplasm. Soluble cargo-protein complexes navigate through the pore by binding to phenylalanine-glycine (FG)-repeat proteins attached to the channel walls. The Nup62 complex contains the FG-repeat proteins Nup62, Nup54, and Nup58 and is located in the center of the NPC. The three proteins bind each other via conserved coiled-coil segments. To determine the stoichiometry of the Nup62 complex, we undertook an in vitro study using gel filtration and analytical ultracentrifugation. Our results reveal a 1:1:1 stoichiometry of the Nup62 complex, where Nup54 is central with direct binding to Nup62 and Nup58. At high protein concentration, the complex forms larger assemblies while maintaining the Nup62:Nup54:Nup58 ratio. For the homologous Nsp1 complex from Saccharomyces cerevisiae, we determine the same stoichiometry, indicating evolutionary conservation. Furthermore, we observe that eliminating one binding partner can result in the formation of complexes with noncanonical stoichiometry, presumably because unpaired coiled-coil elements tend to find a promiscuous binding partner. We suggest that these noncanonical stoichiometries observed in vitro are unlikely to be physiologically relevant.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 Institut für Chemie und Biochemie, AG Strukturbiochemie, Freie Universität Berlin, 14195 Berlin, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Ulrich
authors.forename: James R
authors.initials: JR
authors.lastname: Partridge
authors.forename: Thomas U
authors.initials: TU
authors.lastname: Schwartz
mesh_terms: D000818:Animals; D008562:Membrane Glycoproteins / Q000737:chemistry*; D022022:Nuclear Pore / Q000648:ultrastructure; D028861:Nuclear Pore Complex Proteins / Q000737:chemistry*; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D051381:Rats; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D012996:Solutions
chemical_list: D008562:Membrane Glycoproteins; D028861:Nuclear Pore Complex Proteins; C582177:Nup54 protein, rat; C518579:Nup58 protein, rat; D029701:Saccharomyces cerevisiae Proteins; D012996:Solutions; C049312:nuclear pore protein p62