In situ structural analysis of the human nuclear pore complex

Abstract

SummaryNuclear pore complexes (NPCs) are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Elucidating their 110 MDa structure imposes a formidable challenge and requires in situ structural biology approaches. Fifteen out of about thirty nucleoporins (Nups) are structured and form the Y- and inner ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ∼60 nm in diameter 1. The scaffold is decorated with transport channel Nups that often contain phenylalanine (FG)-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y-complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here, we combined cryo electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modeling to generate the most comprehensive architectural model of the NPC to date. Our data suggest previously unknown protein interfaces across Y-complexes and to inner ring complex members. We demonstrate that the higher eukaryotic transport channel Nup358 (RanBP2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport channel Nups. We conclude that, similarly to coated vesicles, multiple copies of the same structural building block - although compositionally identical - engage in different local sets of interactions and conformations.

Integrative structure and functional anatomy of a nuclear pore complex

“…5D). The inner ring is anchored to the pore membrane through membrane-binding motifs (MBMs) on the β-propellers at the N-termini of Nup157 and Nup170, and on the C-termini of Nup53 and Nup59 17–19 . These proteins also interact with the scaffold-facing domains of Pom152, Ndc1, and Pom34, each of which carry transmembrane domains (TMDs) (Fig.…”

“…In yeast, each outer ring is formed by 8 copies of the Nup84 complex (Figs. 3F, 4), whereas in vertebrates each outer ring contains 16 copies of the equivalent Y-shaped complex arranged in two interlocked rings 17,40 . Moreover, we see neither an additional copy of Nup157/Nup170 connecting the outer and inner rings nor Nup188/Nup192 in the outer rings, as indicated in humans 6 (Fig.…”

“…Fourth, the structure was also validated by its comparison to the core scaffold maps of the Homo sapiens NPC, based primarily on EM density maps 79,80,140,141 (Extended Data Fig. 10).…”

Comparative interactomics provides evidence for functional specialization of the nuclear pore complex

“…89 variations of the outer ring complex in opisthokonts have revealed an interesting architectural divergence between yeast and metazoa. The most compelling example is the metazoan outer ring, which despite near identical compositional and architectural similarity with yeast, comprises 2 reticulated rings, 11,21 in contrast to a single ring in yeast. 8,15,22 Thus, architectural and functional similarities cannot be assumed simply from a similar list of components.…”

“…1). [3][4][5][6][7][8][9][10][11] Poms and the core scaffold form the major structural framework while FG-Nups establish the permeability barrier of the NPC and facilitate nucleocytoplasmic transport through interactions with soluble transport factors (karyopherins), with directionality dependent on a Ran GTP/GDP gradient. 12 There is extremely low sequence similarity between excavate and opisthokont Nups.…”