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

doi:10.1016/S1097-2765(03)00116-3

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 journal › Article › peer-review

231 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 language	English
Pages (from-to)	853-864
Number of pages	12
Journal	Molecular Cell
Volume	11
Issue number	4
DOIs	https://doi.org/10.1016/S1097-2765(03)00116-3
State	Published - 1 Apr 2003
Externally published	Yes

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.

Funding

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.

Funders	Funder number
Marcus Sieff Foundation
National Institutes of Health
National Institute of General Medical Sciences	R01GM033279
United States-Israel Binational Science Foundation

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title = "Removal of a single pore subcomplex results in vertebrate nuclei devoid of nuclear pores",

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.",

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AU - Harel, Amnon

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AU - Shah, Sundeep

AU - Zimmerman, Ella

AU - Elbaum, Michael

AU - Forbes, Douglass J.

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

Abstract

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