Interference with the cytoplasmic tail of gp210 disrupts "close apposition" of nuclear membranes and blocks nuclear pore dilation.
We tested the hypothesis that gp210, an integral membrane protein of nuclear pore complexes (NPCs), mediates nuclear pore formation. Gp210 has a large lumenal domain and small COOH-terminal tail exposed to the cytoplasm. We studied the exposed tail. We added recombinant tail polypeptides to Xenopus nuclear assembly extracts, or inhibited endogenous gp210 tails using anti-tail antibodies. Both strategies had no effect on the formation of fused flattened nuclear membranes, but blocked NPC assembly and nuclear growth. Inhibited nuclei accumulated gp210 and some nucleoporin p62, but failed to incorporate nup214/CAN, nup153, or nup98 and were defective for nuclear import of lamin B3. Scanning and transmission EM revealed a lack of "closely apposed" inner and outer membranes, and the accumulation of novel arrested structures including "mini-pores." We conclude that gp210 has early roles in nuclear pore formation, and that pore dilation is mediated by gp210 and its tail-binding partner(s). We propose that membrane fusion and pore dilation are coupled, acting as a mechanism to control nuclear pore size.
Drummond
Sheona P
SP
Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Wilson
Katherine L
KL
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000378:metabolism; D015151:Immunoblotting; D007381:Intermediate Filament Proteins / Q000378:metabolism; D034921:Lamin Type B; D008561:Membrane Fusion; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011874:Radiometry; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D014981:Xenopus; D029867:Xenopus Proteins
D018076:DNA, Complementary; D007381:Intermediate Filament Proteins; D034921:Lamin Type B; D008562:Membrane Glycoproteins; C512903:NUP210 protein, Xenopus; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; D005982:Glutathione Transferase; D013917:Thrombin