Figure 1. Steady state analysis of nuclear protein import using transfection. Using chemical (e.g. liposome-mediated) or physical (e.g. electroporation) methods, cells can be induced to take up plasmid DNA that encodes the protein to be analysed. After 1–2 days to allow protein expression and nuclear translocation, the cells are fixed and subcellular localization of the protein of interest determined by immunofluorescence and fluorescence microscopy. Results (bottom) are shown for cells transfected in parallel expressing either an NLS-containing protein (left) or an NLS mutant variant (right).

Figure 2. Reconstitution of signal-dependent nuclear protein import in vitro. Since the nuclear envelope has a lipid composition distinct from that of the plasma membrane and in particular is low in cholesterol, the detergent digitonin can be used to permeabilize the plasma membrane and leave the nuclear envelope intact (Adam et al., 1990). Nuclear import of NLS-containing proteins can then be reconstituted through adding cytosolic factors and an ATP-regenerating system. Results (bottom) are shown for 2 and 40 minute time-points of accumulation of an NLS-containing protein.

Figure 3. Kinetic analysis of the regulation of NLS-dependent nuclear protein import. The TF SWI5 is normally only present in the yeast nucleus in G₁ of the cell cycle as the result of cell cycle-dependent phosphorylation by CDC28 kinase. Plots are shown from an experiment in which fluorescently labelled wild-type SWI5 fusion protein able to be phosphorylated by CDC28, and a phosphorylation site mutant variant were microinjected into HTC rat hepatoma cells and transport kinetics were measured using confocal laser scanning microscopy and image analysis. Only in the case of the mutant, where CDC28 phosphorylation is absent, is nuclear accumulation observed, to levels about 8-fold those in the cytoplasm. See Jans DA et al. (1995). Insets show confocal micrographs 40 minutes after microinjection.