Long Unfolded Linkers Facilitate Membrane Protein Import Through the Nuclear Pore Complex

Meinema, Anne C; Laba, Justyna K; Hapsari, Rizqiya Astri; Otten, Renee; Mulder, Frans A. A.; Kralt, Annemarie; Bogaart, Geert van den; Lusk, C. Patrick; Poolman, Bert; Veenhoff, Liesbeth M.

doi:10.1126/science.1205741

Cited by 131 publications

(202 citation statements)

References 22 publications

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“…5C) that could flex to form lateral openings between spokes. This juxtaposition of arches and transient openings may delineate conduits for nucleocytoplasmic transport of transmembrane proteins 23 , potentially resolving the issue of how membrane proteins transit the NPC 24 .…”

Section: Resultsmentioning

confidence: 99%

Integrative structure and functional anatomy of a nuclear pore complex

Kim

Fernández-Martı́nez

Nudelman

et al. 2018

Nature

490

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%

Integrative structure and functional anatomy of a nuclear pore complex

Kim

Fernández-Martı́nez

Nudelman

et al. 2018

Nature

490

894

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“…5B,C), the interaction between endogenous Pom33 and Kap123 is unlikely to be dissociated by Ran-GTP. Indeed, NLSdriven translocation of integral membrane proteins through the nuclear pore membrane has been shown to require a long (at least 120 residues) disordered linker that permits the translocation of NLS-bound karyopherins through the central channel of the NPC (Meinema et al, 2011). Based on predicted topologies (Chadrin et al, 2010; Fig.…”

Section: Discussionmentioning

confidence: 99%

Nuclear pore targeting of the yeast Pom33 nucleoporin depends on karyopherin- and lipid-binding

Floch

Tareste

Fuchs

et al. 2014

Journal of Cell Science

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Pom33 is an integral membrane protein of the yeast nuclear pore complex (NPC), required for proper NPC distribution and assembly. To characterize Pom33 NPC-targeting determinants, we performed immunoprecipitation experiments followed by mass spectrometry analyses. This identified a novel Pom33 partner, the nuclear import factor Kap123. In vitro experiments revealed a direct interaction between Pom33 C-terminal domain (CTD) and Kap123. In silico analysis predicted the presence of two amphipathic α-helices within Pom33-CTD. Circular dichroism and liposome co-flotation assays showed that this domain is able to fold into α-helices in the presence of liposomes and preferentially binds to highly curved lipid membranes. When expressed in yeast, under conditions abolishing Pom33-CTD membrane association, this domain behaves as a Kap123-dependent nuclear localization signal (NLS). While deletion of Pom33 C-terminal domain (Pom33ΔCTD-GFP) impairs Pom33 stability and NPC targeting, mutants affecting either Kap123 binding or the amphipathic properties of the α-helices do not display any detectable defect. However, combined impairment of lipid and Kap123 binding affects Pom33 targeting to NPCs. These data highlight the requirement of multiple determinants and mechanisms for proper NPC localization of Pom33.

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“…45 The NLSs of Heh1 and Heh2 are spaced from the transmembrane domain (TM) by a 180 or 235 amino acid long intrinsically disordered linker (L). The h2NLS together with the long linker is a transplantable signal that conveys INM accumulation to a synthetic transmembrane segment and also to the normally ER-localized Sec61.…”

Section: Kap60/95 Dependent Transport Of Gfp-reportersmentioning

confidence: 99%

“…This proposed transport mechanism of the Heh1 and Heh2-derived proteins resembles that of soluble proteins: it requires RanGTP, Kaps and FG-Nups, and the NLS-encoding domain passes through the central channel. 35,45 A number of observations lead us to think that it is very unlikely that the reporters are transported though the NPC as soluble proteins. While polytopic membrane of these membrane proteins where the Kap60/95 bound h2NLS would pass through the central channel interacting with the FG-Nups while the transmembrane segments diffuse through the pore membrane (Fig.…”

Section: Mechanisms In Addition To Diffusion-retentionmentioning

confidence: 99%

“…First, trapping the reporter at the NPC protein, Nsp1, within the NPC reduces nuclear transport of membrane proteins but not that of soluble proteins. Also, the translocation of h2NLS-L-TM through the NPC is disrupted in a strain that lacks the NPC protein Nup170, 45 similar as Heh2, 35 while the transport of soluble cargo h2NLS-GFP is not affected (Fig. 3; ref.…”

Section: Implications For the Structure Of The Npcmentioning

confidence: 99%

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