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Inner/Outer nuclear membrane fusion in nuclear pore assembly: biochemical demonstration and molecular analysis

Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and...

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Published in:Molecular biology of the cell 2010-12, Vol.21 (23), p.4197-4211
Main Authors: Fichtman, Boris, Ramos, Corinne, Rasala, Beth, Harel, Amnon, Forbes, Douglass J
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Language:English
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cited_by cdi_FETCH-LOGICAL-c351t-2a31b1f82333b3571c32b699041e0485ed6c4ec0209969b762c513238a7bde003
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container_issue 23
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container_title Molecular biology of the cell
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creator Fichtman, Boris
Ramos, Corinne
Rasala, Beth
Harel, Amnon
Forbes, Douglass J
description Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel has little evolutionary precedent. Here we mapped inner/outer nuclear membrane fusion in NPC assembly biochemically by using novel assembly intermediates and membrane fusion inhibitors. Incubation of a Xenopus in vitro nuclear assembly system at 14°C revealed an early pore intermediate where nucleoporin subunits POM121 and the Nup107-160 complex were organized in a punctate pattern on the inner nuclear membrane. With time, this intermediate progressed to diffusion channel formation and finally to complete nuclear pore assembly. Correct channel formation was blocked by the hemifusion inhibitor lysophosphatidylcholine (LPC), but not if a complementary-shaped lipid, oleic acid (OA), was simultaneously added, as determined with a novel fluorescent dextran-quenching assay. Importantly, recruitment of the bulk of FG nucleoporins, characteristic of mature nuclear pores, was not observed before diffusion channel formation and was prevented by LPC or OA, but not by LPC+OA. These results map the crucial inner/outer nuclear membrane fusion event of NPC assembly downstream of POM121/Nup107-160 complex interaction and upstream or at the time of FG nucleoporin recruitment.
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subjects Active Transport, Cell Nucleus
Animals
Cell Nucleus - metabolism
Fluorescent Antibody Technique
Ion Channels - antagonists & inhibitors
Lysophosphatidylcholines - pharmacology
Membrane Fusion
Nuclear Envelope - metabolism
Nuclear Pore Complex Proteins - metabolism
Nuclear Proteins - metabolism
Oleic Acid - pharmacology
Temperature
Xenopus
title Inner/Outer nuclear membrane fusion in nuclear pore assembly: biochemical demonstration and molecular analysis
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