In VivoAnalysis of Nuclear Protein Traffic in Mammalian Cells (original) (raw)

The nuclear pore complex also mediates diffusion, We have developed an experimental system to study and proteins of molecular masses of 40 kDa or less, nucleocytoplasmic traffic of proteins in living mamma-exemplified by the catalytic subunit of protein kinase lian cells. Toward this goal, substrates were generated A, can enter the nucleus via passive diffusion . By that contain several copies of Aequorea victoria green contrast, proteins that exceed the size of the diffusion fluorescent protein (GFP). To follow facilitated transchannel enter the nucleus by a facilitated process that port across the nuclear envelope we created reporter requires energy and a NLS. Polypeptides larger than proteins that carry different nuclear localization se-70 kDa are excluded from nuclei if they do not carry a quences (NLSs). The expression of reporter genes was functional NLS . NLSs can be divided into several controlled by an inducible promoter. Transiently classes. The NLS present in SV40 T-antigen is an extransfected HeLa cells were employed to follow the ample of a ''simple'' NLS containing a stretch of basic sorting of fluorescent reporter proteins. When NLSamino acid residues. A second class of NLSs contains GFP fusions were located in HeLa cells, we found that ''bipartite'' sequences that carry two clusters of posidirect fusion of the NLS derived from SV40 T-antigen tively charged amino acid residues that are separated to GFP prevented nuclear accumulation of the protein. by a spacer region, such as the NLS of Xenopus laevis However, insertion between NLS and GFP of different nucleoplasmin. Another type of targeting sequence inlinkers encoding small amino acid residues produced cludes complex NLSs which do not fit into the classes reporter proteins that were competent for nuclear imof simple or bipartite signals.