Fig. 4. Demonstration that EXP is an LMB-sensitive NES. The EXP region (residues 505-554) of Dd-STATc was inserted at the N terminus of GFP, to yield EXP:GFP, under transcriptional control of the Actin 15 promoter. This construct contains a G418 resistance cassette and was transformed into Dictyostelium. Clones of expressing cells containing multiple copies of EXP:GFP were selected using G418. GFP diffuses freely into the nucleus but in EXP:GFP-transformed cells, here shaken for 3 hours under starvation conditions, the fusion protein is selectively excluded from the nucleus (the positions of the nuclei are indicated by yellow arrows). When, however, cells are incubated for 90 minutes with LMB (20 nM) exclusion is reversed and there is a uniform GFP signal over the cell or, occasionally, even a slight nuclear enrichment [we have previously shown that there is sometimes a low level of nuclear enrichment of GFP itself when it is expressed in Dictyostelium (Ginger et al., 2000)].

Return to article