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.
Zhang
C
C
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia. chunfang.zhang@med.monash.edu.au
Sriratana
A
A
Minamikawa
T
T
Nagley
P
P
D000102:Acetyl-CoA C-Acyltransferase / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000528:radiation effects; D019556:COS Cells / Q000166:cytology; D002470:Cell Survival / Q000528:radiation effects; D003001:Cloning, Molecular; D005456:Fluorescent Dyes / Q000032:analysis; D049452:Green Fluorescent Proteins; D000882:Haplorhini; D006801:Humans; D008027:Light; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D010787:Photosensitivity Disorders; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000102:Acetyl-CoA C-Acyltransferase