Ordered Regions of Channel Nucleoporins Nup62, Nup54, and Nup58 Form Dynamic Complexes in Solution*
Alok Sharma1, Sozanne R. Solmaz12, Günter Blobel3 and Ivo Melčák4
From the Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10065
↵3 An Investigator of the Howard Hughes Medical Institute. To whom correspondence may be addressed: Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065. Tel.: 212-327-8096; Fax: 212-327-7880; E-mail: blobel{at}rockefeller.edu.
↵4 To whom correspondence may be addressed: Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Ave., New York, NY 10065. Tel.: 212-327-8181; Fax: 212-327-7880; E-mail: melcaki{at}rockefeller.edu.
↵1 Both authors contributed equally to this work.
↵2 Present address: Dept. of Chemistry, State University of New York at Binghamton, P. O. Box 6000, Binghamton, NY, 13902.
Background: The ordered region (∼150–200 residues) of each channel nucleoporin is subdivided into segments of ∼40–80 residues.
Results: In solution, ordered regions associate into dynamic and heterogeneous complexes utilizing previously identified interactions between cognate segments.
Conclusion: Solution data are consistent with channel model reconstructed from crystal structures of cognate segments.
Significance: Data support the “ring cycle” model for dilation and constriction of the nuclear pore channel.
Abstract
Three out of ∼30 nucleoporins, Nup62, Nup54, and Nup58, line the nuclear pore channel. These “channel” nucleoporins each contain an ordered region of ∼150–200 residues, which is predicted to be segmented into 3–4 α-helical regions of ∼40–80 residues. Notably, these segmentations are evolutionarily conserved between uni- and multicellular eukaryotes. Strikingly, the boundaries of these segments match our previously reported mapping and crystal data, which collectively identified two “cognate” segments of Nup54, each interacting with cognate segments, one in Nup58 and the other one in Nup62. Because Nup54 and Nup58 cognate segments form crystallographic hetero- or homo-oligomers, we proposed that these oligomers associate into inter-convertible “mid-plane” rings: a single large ring (40–50 nm diameter, consisting of eight hetero-dodecamers) or three small rings (10–20 nm diameter, each comprising eight homo-tetramers). Each “ring cycle” would recapitulate “dilation” and “constriction” of the nuclear pore complex's central transport channel. As for the Nup54·Nup62 interactome, it forms a 1:2 triple helix (“finger”), multiples of which project alternately up and down from mid-plane ring(s). Collectively, our previous crystal data suggested a copy number of 128, 64, and 32 for Nup62, Nup54, and Nup58, respectively, that is, a 4:2:1 stoichiometry. Here, we carried out solution analysis utilizing the entire ordered regions of Nup62, Nup54, and Nup58, and demonstrate that they form a dynamic “triple complex” that is heterogeneously formed from our previously characterized Nup54·Nup58 and Nup54·Nup62 interactomes. These data are consistent both with our crystal structure-deduced copy numbers and stoichiometries and also with our ring cycle model for structure and dynamics of the nuclear pore channel.
Key words: biophysics cell biology nuclear pore nuclear transport protein assembly FG nucleoporins Light scattering Nuclear Pore Complex Ring cycle hypothesis Transport channel
FREE PDF GRATIS: Journal of Biological Chemistry
