Atomic structure of the Y complex of the nuclear pore

Abstract

The nuclear pore complex (NPC) is the principal gateway for transport into and out of the nucleus. Selectivity is achieved through the hydrogel-like core of the NPC. The structural integrity of the NPC depends on ~15 architectural proteins, which are organized in distinct subcomplexes to form the >40-MDa ring-like structure. Here we present the 4.1-Å crystal structure of a heterotetrameric core element ('hub') of the Y complex, the essential NPC building block, from Myceliophthora thermophila. Using the hub structure together with known Y-complex fragments, we built the entire ~0.5-MDa Y complex. Our data reveal that the conserved core of the Y complex has six rather than seven members. Evolutionarily distant Y-complex assemblies share a conserved core that is very similar in shape and dimension, thus suggesting that there are closely related architectural codes for constructing the NPC in all eukaryotes.

Figure 1. Structure of the Myceliophthora thermophila Y-complex hub at 4.1 Å resolution

(a) Schematic of the Y-complex. Regions included in the crystallization construct are colored, other Y-complex regions in grey. Elements of ACE1 fold proteins are indicated: T – tail and C – crown flank the central trunk element. (b) The hub structure is colored as follows: Nup85 (orange), Nup120 (green), Nup145C (cyan), and the Sec13 β-propeller (grey). N and C termini are indicated for the helical proteins. Helices are numbered according to previously solved S. cerevisiae fragments^,,. Numbers that include a letter modifier indicate helical elements not present in S. cerevisiae. (c) Top-down view of the hub. The N terminus of Nup145C is not indicated because it is obscured by the 85° rotation.

Figure 2. Fitness analysis of hub interactions

(a) Growth curves of NUP85Δ strains carrying NUP85:URA3 and either empty pRS315 (negative control), Nup85 wildtype, or Nup85 Δα30 grown in the presence of 5-FOA. The positive control is the NUP85Δ strain carrying NUP85/URA3 and empty pRS315 grown in the absence of 5-FOA. (b) Growth curves of NUP145Δ strains carrying NUP145/URA3 and either empty pRS315 (negative control), Nup145C wildtype, or Nup145C Δα27 grown in the presence of 5-FOA. The positive control is the NUP145Δ strain carrying NUP145/URA3 and empty pRS315 grown in the absence of 5-FOA. (c) The growth curves of NUP120Δ strains carrying YClac33 empty vector (negative control), Nup120 wildtype, or Nup120 Δα30 grown in YPD. Four technical replicates (n=4 OD measurements) for each of three biological replicates (n=3), from separate colonies, were performed at 30 °C for all experiments. All error bars are standard deviation of the mean (s.e.m.).

Figure 3. Composite high-resolution structure of the Y-complex

(a) Composite, hexameric Y-complex core constructed from the hub structure (Fig. 1) combined with previously published X-ray crystal structure fragments. Within Nup84, 4 helices were modeled computationally. (b) Composite S. cerevisiae Y-complex based on A), with S. cerevisiae sequences threaded onto existing homologous structures. Compared to the universally conserved hexameric Y-complex core shown in A), Seh1 (red) is an additional component found in many organisms, including yeast. (c) Composite H. sapiens Y-complex with H. sapiens sequences threaded onto existing homologous structures. Nup37 (blue) is another Y-complex component only found in a subset of eukaryotes, including humans. (d) Space filling surface view of the composite, hexameric Y-complex viewed from the front. (e) Side view. (f) Tilted view.

Figure 4. Comparison of the X-ray based, composite Y-complex structure with published 3-D EM reconstruction structures

(a,b) Electron density envelope around the composite H. sapiens Y-complex calculated for 35 Å resolution from front (a) or top (b) view. (c,d) 3-D EM reconstruction of the H. sapiens Y-complex with an overlay of the composite model, fitted computationally from front (c) or top (d) view. (e,f)Electron density envelope around the composite S. cerevisiae Y-complex calculated at 33 Å resolution from front (e) or top (f) view. (g,h) 3-D EM reconstruction of the S. cerevisiae Y-complex with an overlay of the composite model, fitted computationally from front (g) or top (h) view.

Figure 5. Flexibility of the Y-complex

Experimentally observed hinge regions of the Y-complex are denoted by dashed lines. (a) ref. . (b–c) ref. . (d–e), refs. -,,. (f) ref. .

Figure 6. Fitting of the composite H. sapiens Y-complex into the cryo-ET map of the entire NPC

(a) Consensus map calculated from the cryo-ET map of the human NPC (EMD code: 2444). The cytoplasmic ring density is highlighted in grey for clarity. (b) The top scoring fit of the composite H. sapiens Y-complex (conformation 1) and the second top scoring fit (conformation 2) are depicted. (c) A superposition of the two fits from (b) shows that they are related by a ~20° rotation about the Y-complex hub, and substantial bending of the long stack.