IN-VIVO ANALYSIS OF NUCLEAR-PROTEIN TRAFFIC IN MAMMALIAN-CELLS

We have developed an experimental system to study nucleocytoplasmic tr affic of proteins in Living mammalian cells. Toward this goal, substra tes were generated that contain several copies of Aequorea victoria gr een 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 gen es was controlled by an inducible promoter. Transiently transfected He La cells were employed to follow the sorting of fluorescent reporter p roteins. When NLS-GFP fusions were located in HeLa cells, we found tha t direct fusion of the NLS derived from SV40 T-antigen to GFP prevente d nuclear accumulation of the protein. However, insertion between NLS and GFP of different linkers encoding small amino acid residues produc ed reporter proteins that were competent for nuclear import. Taken tog ether, we have generated unique tools for the characterization of nucl ear protein import in dividing mammalian cells. (C) 1997 Academic Pres s.