Removal of a single pore subcomplex results in vertebrate nuclei devoid of nuclear pores.
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.
Harel
Amnon
A
Section of Cell and Developmental Biology, Division of Biology 0347, University of California, San Diego, La Jolla 92093, USA.
Orjalo
Arturo V
AV
Vincent
Thomas
T
Lachish-Zalait
Aurelie
A
Vasu
Sanjay
S
Shah
Sundeep
S
Zimmerman
Ella
E
Elbaum
Michael
M
Forbes
Douglass J
DJ
D000818:Animals; D005260:Female; D006801:Humans; D021581:Active Transport, Cell Nucleus / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D006367:HeLa Cells; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D015778:Minor Histocompatibility Antigens; D022022:Nuclear Pore / Q000378:metabolism / Q000648:ultrastructure; D028861:Nuclear Pore Complex Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins; D009865:Oocytes / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000172:deficiency / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D014714:Vertebrates / Q000378:metabolism; D014982:Xenopus laevis; D029867:Xenopus Proteins
D008565:Membrane Proteins; D015778:Minor Histocompatibility Antigens; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; C512772:NUP107 protein, human; C440250:NUP133 protein, human; C440251:NUP160 protein, human; C499080:Nup160 protein, Xenopus; C106899:NUP85 protein, S cerevisiae; D010980:Platelet Membrane Glycoproteins; C082264:Pom121 protein, rat; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D029867:Xenopus Proteins; C063865:NUP210 protein, human