In vivo analysis of nuclear protein traffic in mammalian cells.

We have developed an experimental system to study nucleocytoplasmic traffic of proteins in living mammalian cells. Toward this goal, substrates were generated that contain several copies of Aequorea victoria green fluorescent protein (GFP). To follow facilitated transport across the nuclear envelope we created reporter proteins that carry different nuclear localization sequences (NLSs). The expression of reporter genes was controlled by an inducible promoter. Transiently transfected HeLa cells were employed to follow the sorting of fluorescent reporter proteins. When NLS-GFP fusions were located in HeLa cells, we found that direct fusion of the NLS derived from SV40 T-antigen to GFP prevented nuclear accumulation of the protein. However, insertion between NLS and GFP of different linkers encoding small amino acid residues produced reporter proteins that were competent for nuclear import. Taken together, we have generated unique tools for the characterization of nuclear protein import in dividing mammalian cells.