Diffusion of proteins across the nuclear envelope of HeLa cells

We describe an experimental system to study nucleocytoplasmic diffusion of proteins in living HeLa cells. To localize proteins to the nucleus, substrates were created that contain a nuclear localization sequence fused to Aequorea victoria green fluorescent protein (GFP). Transiently and stably transfected HeLa cells were used for these assays. A protein of 29-kDa molecular mass that harbors GFP and the bipartite Xenopus nucleoplasmin nuclear localization sequence (NLS) accumulates efficiently in nuclei of HeLa cells. However, in the absence of active facilitated nuclear import, the reporter protein exits the nucleus and equilibrates between nucleus and cytoplasm. We define different conditions that promote the diffusion of small nuclear proteins across the nuclear envelope of mammalian culture cells. Our results set the stage to analyze the competence of nuclear pore complexes for nucleocytoplasmic diffusion of macromolecules in living cells.