GREEN-FLUORESCENT PROTEIN FUSIONS FOR EFFICIENT CHARACTERIZATION OF NUCLEAR TARGETING

The green-fluorescent protein (GFP) from Aequorea victoria has been sh own to be a convenient and flexible reporter molecule within a variety of eukaryotic systems, including higher plants. It is particularly su ited for applications in vivo, since the mechanism of fluorophore form ation involves an intramolecular autoxidation and does not require exo genous co-factors. Unlike standard histochemical procedures of fixatio n and staining required for analysis of the cellular or tissue-specifi c expression of other popular reporter molecules, such as the beta-glu curonidase (GUS) marker, analysis of GFP can be done in living cells w ith no specific pretreatments. This implies that GFP might also be par ticularly suited for studies of intracellular protein targeting. In th is paper, the use of GUS is compared with that of GFP for the analysis of nuclear targeting in tobacco. A novel oligopeptide motif from a to bacco protein is described which confers nuclear localization of GUS. The use of this oligopeptide and two from potyviral proteins to target GFP to the nucleus is examined. An essential modification of GFP is d escribed, which specifically increases its molecular weight to elimina te its passive penetration into the nucleus. Three examples of the tar geting of these enlarged GFP molecules to the nucleus are illustrated. GFP, in combination with confocal microscopy, offers significant adva ntages over traditional methods of studying nuclear targeting.