INTERACTIONS AND 3-DIMENSIONAL LOCALIZATION OF A GROUP OF NUCLEAR-PORE COMPLEX PROTEINS

We have used antibodies directed against a number of nuclear pore comp lex (NPC) proteins to determine their mutual interactions and location within the three-dimensional structure of the NPC. A monoclonal antib ody, termed QE5, recognized three NPC polypeptides, p250, NUP153, and p62 on Western blots, and labeled the nuclear envelope of several cult ured cell lines by immunofluorescence microscopy. These three polypept ides contained O-linked N-acetylglucosamine residues and were released from the NPC by detergent/high-salt treatment as discrete high molecu lar weight complexes. p250 was found in association with a novel 75 kD protein, NUP153 was released as a homo-oligomer of about 1 megadalton , and p62 was associated with polypeptides of 58 and 54 kD (previously reported by Finlay, D. R., E. Meier, P. Bradley, J. Horecka, and D. J . Forbes. 1991. J. Cell Biol. 114:169-183). p75, p58, and p54 were not galactosylated in vitro. Xenopus oocyte NEs were labeled with gold-co njugated QE5 and prepared for electron microscopy by quick freezing/fr eeze drying/rotary metal shadowing. This EM preparation method enabled us to more precisely localize the epitopes of this antibody to the cy toplasmic filaments and the nuclear basket of the NPC. Since QE5 recog nizes three O-linked NPC glycoproteins, its labeling was compared with that of the lectin wheat germ agglutinin which recognizes O-linked N- acetylglucosamine moieties. The two probes were found to yield similar , although not identical, distributions of label. To identify the indi vidual proteins with particular NPC components, we have used an anti-p eptide antibody against NUP153 and a monospecific anti-p250 polyclonal antibody. Labeling with these two antibodies has documented that NUP1 53 is a constituent of the nuclear basket with at least one of its epi topes residing in its terminal ring, whereas p250 is a constituent of the cytoplasmic filaments.