Inner/outer nuclear membrane fusion in nuclear pore assembly: Biochemical demonstration and molecular analysis

Boris Fichtman, Corinne Ramos, Beth Rasala, Amnon Harel, Douglass J. Forbes

Research output: Contribution to journalArticlepeer-review

40 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)4197-4211
Number of pages15
JournalMolecular Biology of the Cell
Volume21
Issue number23
DOIs
StatePublished - 1 Dec 2010
Externally publishedYes

Funding

FundersFunder number
National Institute of General Medical SciencesR01GM033279

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