INHIBITION OF NUCLEAR-PROTEIN IMPORT BY A MONOCLONAL-ANTIBODY AGAINSTA NOVEL CLASS OF NUCLEAR-PORE PROTEINS

Nuclear import of proteins is mediated by the nuclear pore complexes i n the nuclear envelope and requires the presence of a nuclear localiza tion signal (NLS) on the karyophilic protein. In this paper, we descri be studies with a monoclonal antibody, Mab E2, which recognizes a clas s of nuclear pore proteins of 60-76 kDa with a common phosphorylated e pitope on rat nuclear envelopes. The Mab Ea-reactive proteins fraction ated with the relatively insoluble pore complex-containing component o f the envelope and gave a finely punctate pattern of nuclear staining in immunofluorescence assays. The antibody did not bind to any cytosol ic proteins. Mab E2 inhibited the interaction of a simian virus 40 lar ge T antigen NLS peptide with a specific 60-kDa NLS-binding protein fr om rat nuclear envelopes in photoaffinity labeling experiments. The an tibody blocked the nuclear import of NLS-albumin conjugates in an in v itro nuclear transport assay with digitonin-permeabilized cells, but d id not affect passive diffusion of a small nonnuclear protein, lysozym e, across the pore. Mab E2 may inhibit protein transport by directly i nteracting with the 60-kDa NLS-binding protein, thereby blocking signa l-mediated nuclear import across the nuclear pore complex. (C) 1994 Ac ademic Press, Inc.