Removal of a single pore subcomplex results in vertebrate nuclei devoid of nuclear pores

Amnon Harel, Arturo V. Orjalo, Thomas Vincent, Aurelie Lachish-Zalait, Sanjay Vasu, Sundeep Shah, Ella Zimmerman, Michael Elbaum, Douglass J. Forbes

Research output: Contribution to journalArticlepeer-review

227 Scopus citations

Abstract

The vertebrate nuclear pore complex, 30 times the size of a ribosome, assembles from a library of soluble subunits and two membrane proteins. Using immunodepletion of Xenopus nuclear reconstitution extracts, it has previously been possible to assemble nuclei lacking pore subunits tied to protein import, export, or mRNA export. However, these altered pores all still possessed the bulk of pore structure. Here, we immunodeplete a single subunit, the Nup107-160 complex, using antibodies to Nup85 and Nup133, two of its components. The resulting reconstituted nuclei are severely defective for NLS import and DNA replication. Strikingly, they show a profound defect for every tested nucleoporin. Even the integral membrane proteins POM121 and gp210 are absent or unorganized. Scanning electron microscopy reveals pore-free nuclei, while addback of the Nup107-160 complex restores functional pores. We conclude that the Nup107-160 complex is a pivotal determinant for vertebrate nuclear pore complex assembly.

Original languageEnglish
Pages (from-to)853-864
Number of pages12
JournalMolecular Cell
Volume11
Issue number4
DOIs
StatePublished - 1 Apr 2003
Externally publishedYes

Bibliographical note

Funding Information:
The authors thank S. Allen for sequence searches, V. Delmar, M. Aviguetero, Zhongsheng You, and K. Wilson of U.C.S.D. for help with experimental techniques, and R. Chan for use of her gp210 antibody prior to publication. M.E. thanks Terry Allen for generous instruction in FESEM. This work was funded by a grant from the NIH (RO1 GM33279) to D.J.F., a United States-Israel Binational Science Foundation grant to M.E. and D.J.F., funding from the Marcus Sieff Foundation to M.E., and GANN Predoctoral Fellowships to A.V.O. and T.V.

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