Regulation of nucleocytoplasmic localization of the atDjC6 chaperone protein

Summary. The sequence of the atDjC6 chaperone protein includes three potential nuclear localization signal (NLS) sequences (A–C) and three potential nuclear export signal (NES) sequences (X–Z). The subcellular localization of atDjC6 was studied by scanning laser confocal microscopy of chimera with the green-fluorescent protein (GFP) transiently expressed in tobacco BY-2 cells. The localization of the atDjC6::GFP chimera was coincident with that of the nuclear stain propidium iodide. Site-directed mutagenesis was used to verify the predicted NLS sequences. Each was individually fused to GFP and tested for protein localization. The individual NLS sequences were sufficient to direct partial nuclear localization of GFP, although the targeting information within NLS-B is apparently conformation sensitive. Site-directed mutagenesis of the NES sequences increased the amount of each chimera that was nuclearly localized, indicating a decrease in nuclear export. When any pair of NLS sequences were appended to GFP, the chimera were entirely nuclearly localized. Quantitative two-hybrid analysis was used to verify that the decoding of NLS sequence information involves interaction with the NLS-receptor protein importin-. Each of the NLS sequences is flanked by a site of potential Ser phosphorylation, and recombinant atDjC6 could be phosphorylated in vitro. Mutagenesis of Ser residues to the P-Ser mimic Asp interfered with nuclear targeting, apparently by preventing recognition or binding by importin-. Our results are consistent with a regulated nucleocytoplasmic localization of the atDjC6 chaperone protein.Correspondence and reprints: Plant Genetics Research Unit, USDA Agricultural Research Service, 108 Curtis Hall, University of Missouri, Columbia, MO 65211, U.S.A.