Structural and functional analysis of Nup120 suggests ring formation of the Nup84 complex - PubMed (original) (raw)

Structural and functional analysis of Nup120 suggests ring formation of the Nup84 complex

Hyuk-Soo Seo et al. Proc Natl Acad Sci U S A. 2009.

Abstract

The Nup84 complex constitutes a key building block in the nuclear pore complex (NPC). Here we present the crystal structure of one of its 7 components, Nup120, which reveals a beta propeller and an alpha-helical domain representing a novel fold. We discovered a previously unidentified interaction of Nup120 with Nup133 and confirmed the physiological relevance in vivo. As mapping of the individual components in the Nup84 complex places Nup120 and Nup133 at opposite ends of the heptamer, our findings indicate a head-to-tail arrangement of elongated Nup84 complexes into a ring structure, consistent with a fence-like coat for the nuclear pore membrane. The attachment site for Nup133 lies at the very end of an extended unstructured region, which allows for flexibility in the diameter of the Nup84 complex ring. These results illuminate important roles of terminal unstructured segments in nucleoporins for the architecture, function, and assembly of the NPC.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Structure of the S. cerevisiae Nup120 NTD. (A) Domain structure. Yellow, β propeller domain; blue, α-helical insertion in the 6D7A loop; green, α-helical domain; gray, α-helical region. The bar above the domain structure denotes the crystallized fragment. (B) Structure of the Nup120 NTD in ribbon representation, color-coded as in (A). A 90°-rotated view is shown on the right.

Fig. 2.

Fig. 2.

Structural analysis of the Nup120 NTD domains. (A) Ribbon representation of the Nup120 β propeller domain. The 7 blades of the β propeller core (yellow), the location of the disordered 1E1A loop (orange), the 3D4A loop (red), the α-helical insertion in the 6D7A loop (blue), and their secondary structure elements are indicated. (B) Schematic representation of the Nup120 β propeller domain and the locations of its various insertions. (C) Ribbon representation of the Nup120 α-helical domain. The leucine zipper–like core (orange) and the 9 surrounding α-helices (green) are indicated. A 180°-rotated view is shown on the right.

Fig. 3.

Fig. 3.

Nup120 NTD interacts with the 15 N-terminal residues of Nup133. (A) Gel filtration profiles of Nup120 NTD (blue), Nup133 NTD (red), and the Nup120 NTD·Nup133 NTD complex (green). (B) Gel filtration profiles of Nup120 NTD (blue), the 15 N-terminal residues of Nup133 fused to GST (red), and their complex (green). All proteins were injected at approximately the same concentrations. (C) The invariant Asp-641 of Nup120 and Arg-11 of Nup133 are key residues for complex formation. The location of Asp-641 and Arg-11 are indicated by asterisks in multispecies sequence alignments. (D) Gel filtration profiles of Nup120 NTD D641A mutant (blue) and the Nup133 NTD (red), and the elution profile resulting from incubation of the 2 proteins before injection (green). (E) Gel filtration profiles of Nup120 NTD (blue) and the Nup133 NTD R11A mutant (red), and the elution profile resulting from incubation of the 2 proteins before injection (green). As a reference, the gel filtration profile of the wild-type Nup120 NTD·Nup133 NTD is indicated in black.

Fig. 4.

Fig. 4.

Physiological relevance of the Nup120–Nup133 interaction. (A) Detection of poly(A)+ mRNA using an Alexa-647 labeled 50-mer oligo dT FISH probe (Top). Wild-type cells (Left) display a diffuse FISH signal, while _nup120_Δ (Middle) and _nup133_Δ (Right) cells yield strong nuclear signals that coincide with DAPI staining, consistent with poly(A)+ mRNA retention inside the nucleus. (B) Quantitation of nuclear poly(A)+ mRNA retention in _nup120_Δ and _nup133_Δ yeast strains complemented with various Nup120 and Nup133 variants. The percentages refer to the fraction of cells that displayed marked nuclear staining and are derived from 3 independent experiments.

Fig. 5.

Fig. 5.

Model for the ring formation of the Nup84 complex. (A) Schematic representation of the heptameric complex and the approximate localization of its 7 nups (19). (B) The interaction of Nup120 and Nup133 suggests the intermolecular interaction between 2 heptamers in a head-to-tail fashion mediated by a short stretch at the very N terminus of an extended unstructured region of Nup133. (C) Eight heptamers are arranged in a closed ring with a diameter of ≈1,000 Å in accordance with the NPC dimensions determined by cryo-electron microscopy (8).

Similar articles

Cited by

References

    1. Hoelz A, Blobel G. Cell biology: Popping out of the nucleus. Nature. 2004;432:815–816. - PubMed
    1. Stewart M. Molecular mechanism of the nuclear protein import cycle. Nat Rev Mol Cell Biol. 2007;8:195–208. - PubMed
    1. Stewart M. Ratcheting mRNA out of the nucleus. Mol Cell. 2007;25:327–330. - PubMed
    1. Reichelt R, et al. Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components. J Cell Biol. 1990;110:883–894. - PMC - PubMed
    1. Beck M, Lucić V, Förster F, Baumeister W, Medalia O. Snapshots of nuclear pore complexes in action captured by cryo-electron tomography. Nature. 2007;449:611–615. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources