VISUALIZATION OF MITOCHONDRIAL PROTEIN IMPORT IN CULTURED-MAMMALIAN-CELLS WITH GREEN FLUORESCENT PROTEIN AND EFFECTS OF OVEREXPRESSION OF THE HUMAN IMPORT RECEPTOR TOM20

The presequence of the ornithine transcarbamylase precursor (pOTC) was fused to green fluorescent protein (GFP), yielding pOTC-GFP and pOTCN -GFP containing the presequence plus 4 and 58 residues of mature ornit hine transcarbamylase, respectively, When GFP cDNA was transfected int o COS-7 cells, the cytosol and nucleus were fluorescent, On the other hand, pOTC-GFP cDNA gave strong fluorescence of a unique mitochondrial pattern, After fractionation of cells expressing pOTC-GFP with digito nin, fluorescence was recovered mostly in the particulate fraction, Im munoblot analysis showed that processed GFP was present in the particu late fraction, whereas pOTC-GFP was recovered in both the soluble and particulate fractions. pOTC-GFP and pOTCN-GFP synthesized in vitro wer e imported efficiently into the isolated mitochondria, Single and trip le amino acid mutations in the presequence resulted in impaired mitoch ondrial import and in a loss of mitochondrial fluorescence. Perinuclea r aggregation of fluorescent mitochondria was observed when the human mitochondrial import receptor Tom20 (hTom20) was coexpressed with pOTC -GFP. Overexpression of hTom20 (not Delta hTom20, which lacks the anch or sequence) resulted in stimulated mitochondrial import of pOTC-GFP i n COS-7 cells, When pOTC-GFP cDNA was microinjected into nuclei of hum an fibroblast cells, mitochondrial fluorescence was detected as early as 2-3 h after injection. These results show that GFP fusion protein c an be used to visualize mitochondrial structures and to monitor mitoch ondrial protein import in a single cell in real time.