Structure–function mapping of a heptameric module in the nuclear pore complex

Fernández-Martı́nez, Javier; Phillips, Jeremy; Sekedat, Matthew D.; Díaz-Avalos, Rubén; Velázquez-Muriel, Javier; Franke, Josef D.; Williams, Rosemary; Stokes, David L.; Chait, Brian T.; Šali, Andrej; Rout, Michael P.

doi:10.1083/jcb.201109008

Cited by 115 publications

(199 citation statements)

References 88 publications

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“…The fitness defects of strains containing Nup truncations provide an estimate of the structural importance of the truncated regions 9,13 . Thus, we quantified the fitness defect of strains containing systematic truncations of every major symmetric Nup using ODELAY 14 (an automated phenotypic analysis platform; Extended Data Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For stochastic sampling methods, thoroughness of sampling can be assessed by showing that multiple independent runs ( e.g ., using random starting configurations and different random number generator seeds, as is the case for structure samples 1 and 2) do not result in significantly different structures 42,66,72,97 . We tested the similarity between structure samples 1 and 2 in the following two ways.…”

Section: Methodsmentioning

confidence: 91%

“…All S. cerevisiae strains used in this study are listed in Supplementary Table 5, with the exception of the Nup84 complex truncation mutants 42 and the Pom152 truncation mutants 43 . Unless otherwise stated, strains were grown at 30°C in YPD media (1% yeast extract, 2% bactopeptone, and 2% glucose).…”

Section: Methodsmentioning

confidence: 99%

“…Strains for which truncations in a haploid background were found to lead to lethality after tetrad dissection (Nic96 and Nup192) were assigned the maximum level of defect and plotted on top of the rest (diploid), based on the fitness phenotype observed for the indicated Nic96 and Nup192 mutants in a diploid background (where a wild type copy of the nucleoporin is also present and expressed). Six divisions were assigned based on decreasing level of fitness 171,175 (white = wild type, to dark purple = severe defect). AU, arbitrary unit; Error = standard deviation.…”

Section: Extended Datamentioning

confidence: 99%

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Integrative structure and functional anatomy of a nuclear pore complex

Kim

Fernández-Martı́nez

Nudelman

et al. 2018

Nature

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478

894

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Summary Despite the central role of Nuclear Pore Complexes (NPCs) as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm, their large size and dynamic nature have impeded a full structural and functional elucidation. Here, we have determined a subnanometer precision structure for the entire 552-protein yeast NPC by satisfying diverse data including stoichiometry, a cryo-electron tomography map, and chemical cross-links. The structure reveals the NPC’s functional elements in unprecedented detail. The NPC is built of sturdy diagonal columns to which are attached connector cables, imbuing both strength and flexibility, while tying together all other elements of the NPC, including membrane-interacting regions and RNA processing platforms. Inwardly-directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized in distinct functional units. Taken together, this integrative structure allows us to rationalize the architecture, transport mechanism, and evolutionary origins of the NPC.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 91%

Section: Methodsmentioning

confidence: 99%

Section: Extended Datamentioning

confidence: 99%

See 2 more Smart Citations

Integrative structure and functional anatomy of a nuclear pore complex

Kim

Fernández-Martı́nez

Nudelman

et al. 2018

Nature

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478

894

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“…We also performed functional analysis of NPC perturbations using ODELAY [5] and heat-mapped these data to interpret the structure of the NPC in a functional context at sub-nanometer precision [6,7].…”

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confidence: 99%

Integrative Structure and Functional Anatomy of a Nuclear Pore Complex

et al. 2018

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Nuclear Pore Complexes (NPCs) are large proteinaceous assemblies studded through the nuclear envelope (NE), the double-membraned barrier surrounding the nucleus; they are the sole mediators of macromolecular transport between the nucleus and the cytoplasm, and carry key regulatory platforms for numerous nuclear processes [1]. NPCs are also major targets for viral manipulation, and defects in this transport machine are directly linked to human diseases, including cancers [2]. Each NPC is an 8-fold symmetric, cylindrical assembly consisting of ~500 copies of ~30 different proteins (nucleoporins or Nups). These Nups assemble into subcomplexes that form higher order structures called spokes. Eight spokes assemble into even larger modules: coaxial outer and inner rings form a symmetric core scaffold, which is connected to a membrane ring, a nuclear basket, and cytoplasmic RNA export complexes [3]. The scaffold surrounds a central channel, which is formed in part by multiple intrinsically disordered Phe-Gly (FG) repeat motifs extending from nucleoporins termed FG Nups. These FG motifs mediate selective nucleocytoplasmic transport through specific interactions with nuclear transport factors (NTRs) carrying their cognate macromolecular cargoes [4].We used an integrative structure determination strategy to determine a subnanometer precision structure for the entire 552-protein, 52.3 MDa yeast NPC by satisfying diverse data including stoichiometry, a cryo-electron tomography map, and chemical cross-links. We also performed functional analysis of NPC perturbations using ODELAY [5] and heat-mapped these data to interpret the structure of the NPC in a functional context at sub-nanometer precision [6,7].We found that at the heart of the inner ring, rigid diagonal columns reinforce the NPC's structural integrity (formed by Nic96 as a keystone and flanked by Nup157 and Nup170). Membrane-binding and transmembrane Nups are strategically placed throughout the core scaffold to stabilize pore membrane

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Puzzling out nuclear pore complex assembly

Penzo,

Palancade

2023

FEBS Letters

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Nuclear pore complexes (NPCs) are sophisticated multiprotein assemblies embedded within the nuclear envelope and controlling the exchanges of molecules between the cytoplasm and the nucleus. In this review, we summarize the mechanisms by which these elaborate complexes are built from their subunits, the nucleoporins, based on our ever‐growing knowledge of NPC structural organization and on the recent identification of additional features of this process. We present the constraints faced during the production of nucleoporins, their gathering into oligomeric complexes, and the formation of NPCs within nuclear envelopes, and review the cellular strategies at play, from co‐translational assembly to the enrolment of a panel of cofactors. Remarkably, the study of NPCs can inform our perception of the biogenesis of multiprotein complexes in general – and vice versa.

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Structure–function mapping of a heptameric module in the nuclear pore complex

Abstract: Integration of EM, protein–protein interaction, and phenotypic data reveals novel insights into the structure and function of the nuclear pore complex’s ∼600-kD heptameric Nup84 complex.

Cited by 115 publications

References 88 publications

Integrative structure and functional anatomy of a nuclear pore complex

Integrative structure and functional anatomy of a nuclear pore complex

Integrative Structure and Functional Anatomy of a Nuclear Pore Complex

Puzzling out nuclear pore complex assembly

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