Photosensitisation properties of mitochondrially localised green fluorescent protein

The photosensitisation properties of a mitochondrially localised green fluorescent protein (GFP) variant were established in cultured monkey kidney cells. We first cloned into a mammalian expression vector the thermostable variant GFP5, fused at its N-terminus to the 16-amino acid mitochondrial targeting sequence of human 3-oxoacyl-CoA thiolase. The recombinant plasmid thus constructed, pCZ34, was transfected into COS7 cells, under conditions for transient expression. GFP5 was shown to have a mitochondrial localisation by confocal microscopy, confirmed by its almost identical pattern with that of MitoTracker Red which selectively stains mitochondria. After photoirradiation of such transfected COS7 cells with 390-570 nm light to excite the mitochondrially localised GFP5, significant cell killing was observed. DAPI-staining of the dead cells detached from the growth support revealed cells to have undergone apoptosis. Thus, GFP5 can be used in the development of an organelle-specific photosensitiser since it can be targeted to different subcellular locations by protein engineering of the signal sequences.