The stoichiometry of the nucleoporin 62 subcomplex of the nuclear pore in solution - PubMed (original) (raw)

The stoichiometry of the nucleoporin 62 subcomplex of the nuclear pore in solution

Alexander Ulrich et al. Mol Biol Cell. 2014 May.

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.

PubMed Disclaimer

Figures

FIGURE 1:

FIGURE 1:

Protein complexes used in this study. Definitions of all Nup58-Nup54-Nup62, Nup58-Nup54, and Nup54-Nup62 complexes used in our study. Narrow tubes represent FG-repeat regions; broad cylinders represent α-helical, predicted coiled-coil domains. Numbers indicate residue positions in the R. norvegicus proteins. Numbers in italics indicate protein residues in the S. cerevisiae homologues. Regions present in the particular complex are colored. The scNup49-Nup57-T4 Lysozyme complex contains, in addition to the residues of the scNup49-Nup57 complex, T4 Lysozyme N-terminally fused to Nup57.

FIGURE 2:

FIGURE 2:

Final purity of protein complexes used in AUC experiments. Size exclusion chromatograms and SDS–PAGE gels of protein complexes used in AUC experiments. Proteins were purified as described in Materials and Methods. Final purity as obtained after gel filtration with S200 10/300 column of (A) Nup58-Nup54-Nup62 complexes, (B) Nup58-Nup54 complexes, and (C) Nup54-Nup62 complexes, shown by size exclusion chromatograms and SDS–PAGE gels. Nup57 autodegradation fragments are indicated with asterisks.

FIGURE 3:

FIGURE 3:

Concentration-dependent oligomerization of Nup58-Nup54-Nup62 complexes. Size exclusion chromatograms and SDS–PAGE gels of a high (5 mg/ml or 3.8 mg/ml) and a low (0.5 mg/ml) concentration of (A) the long rnNup58-Nup54-Nup62 complex, (B) the short rnNup58-Nup54-Nup62 complex, and (C) the scNup49-Nup57-Nsp1 complex. scNup57 autodegradation fragments are indicated by asterisks. Experiments were performed as described in Materials and Methods.

FIGURE 4:

FIGURE 4:

Models of protein subcomplexes of the Nup62/Nsp1 complex. Complex models are based on size exclusion chromatography and AUC data. Cylinders symbolize predicted coiled-coil portions of nucleoporins. Double arrows indicate dynamic exchange between oligomeric states. Cartoon representations of oligomerization behavior and composition of (A) the trimeric complexes, (B) the rnNup54-Nup58/scNup49-Nup57 two-component complexes, and (C) the rnNup54-Nup62/scNup57-Nsp1 two-component complexes. We suggest that complexes depicted in (A) likely represent the complex ensemble in vivo, whereas complexes depicted in (B) and (C) represent assemblies, which, likely artificially, occur in vitro in the absence of the third binding partner.

Similar articles

Cited by

References

    1. Akey CW, Radermacher M. Architecture of the Xenopus nuclear pore complex revealed by three-dimensional cryo-electron microscopy. J Cell Biol. 1993;122:1–19. - PMC - PubMed
    1. Alber F, et al. The molecular architecture of the nuclear pore complex. Nature. 2007;450:695–701. - PubMed
    1. Amlacher S, Sarges P, Flemming D, van Noort V, Kunze R, Devos DP, Arumugam M, Bork P, Hurt E. Insight into structure and assembly of the nuclear pore complex by utilizing the genome of a eukaryotic thermophile. Cell. 2011;146:277–289. - PubMed
    1. Bailer SM, Balduf C, Hurt E. The Nsp1p carboxy-terminal domain is organized into functionally distinct coiled-coil regions required for assembly of nucleoporin subcomplexes and nucleocytoplasmic transport. Mol Cell Biol. 2001;21:7944–7955. - PMC - PubMed
    1. Beck M, Förster F, Ecke M, Plitzko JM, Melchior F, Gerisch G, Baumeister W, Medalia O. Nuclear pore complex structure and dynamics revealed by cryoelectron tomography. Science. 2004;306:1387–1390. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources