PHOTOSENSITIZATION PROPERTIES OF MITOCHONDRIALLY LOCALIZED 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 aci d mitochondrial targeting sequence of human 3-oxoacyl-CoA thiolase. Th e recombinant plasmid thus constructed, pCZ34, was transfected into CO S7 cells, under conditions far transient expression. GFP5 was shown to have a mitochondrial localisation by confocal microscopy, confirmed b y its almost identical pattern with that of MitoTracker Red which sele ctively stains mitochondria. After photoirradiation of such transfecte d COS7 cells with 390-570 nm light to excite the mitochondrially local ised GFP5, significant cell killing was observed. DAPI-staining of the dead cells detached from the growth support revealed cells to have un dergone apoptosis. Thus, GFP5 can be used in the development of an org anelle-specific photosensitiser since it can be targeted to different subcellular locations by protein engineering of the signal sequences. (C) 1998 Academic Press.