Importin beta regulates the seeding of chromatin with initiation sites for nuclear pore assembly.
The nuclear envelope of higher eukaryotic cells reforms at the exit from mitosis, in concert with the assembly of nuclear pore complexes (NPCs). The first step in postmitotic NPC assembly involves the "seeding" of chromatin with ELYS and the Nup107-160 complex. Subsequent steps in the assembly process are poorly understood and different mechanistic models have been proposed to explain the formation of the full supramolecular structure. Here, we show that the initial step of chromatin seeding is negatively regulated by importin beta. Direct imaging of the chromatin attachment sites reveals single sites situated predominantly on the highest substructures of chromatin surface and lacking any sign of annular structures or oligomerized pre-NPCs. Surprisingly, the inhibition by importin beta is only partially reversed by RanGTP. Importin beta forms a high-molecular-weight complex with both ELYS and the Nup107-160 complex in cytosol. We suggest that initiation sites for NPC assembly contain single copies of chromatin-bound ELYS/Nup107-160 and that the lateral oligomerization of these subunits depends on the recruitment of membrane components. We predict that additional regulators, besides importin beta and Ran, may be involved in coordinating the initial seeding of chromatin with subsequent steps in the NPC assembly pathway.
Rotem
Asaf
A
Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
Gruber
Rita
R
Shorer
Hagai
H
Shaulov
Lihi
L
Klein
Eugenia
E
Harel
Amnon
A
D000818:Animals; D002843:Chromatin / Q000378:metabolism* / Q000648:ultrastructure; D002846:Chromatography, Affinity; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D022022:Nuclear Pore / Q000378:metabolism*; D010063:Ovum / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D011485:Protein Binding; D014157:Transcription Factors / Q000378:metabolism; D014981:Xenopus / Q000378:metabolism*; D029867:Xenopus Proteins / Q000378:metabolism; D028961:beta Karyopherins / Q000378:metabolism*; D020931:ran GTP-Binding Protein / Q000378:metabolism
C525081:AHCTF1 protein, Xenopus; D002843:Chromatin; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D029867:Xenopus Proteins; D028961:beta Karyopherins; D020931:ran GTP-Binding Protein