Visualization of Mitochondrial Protein Import in Cultured Mammalian Cells with Green Fluorescent Protein and Effects of Overexpression of the Human Import Receptor Tom20

Abstract

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 ornithine transcarbamylase, respectively. When GFP cDNA was transfected into 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 digitonin, fluorescence was recovered mostly in the particulate fraction. Immunoblot analysis showed that processed GFP was present in the particulate fraction, whereas pOTC-GFP was recovered in both the soluble and particulate fractions. pOTC-GFP and pOTCN-GFP synthesized in vitro were imported efficiently into the isolated mitochondria. Single and triple amino acid mutations in the presequence resulted in impaired mitochondrial import and in a loss of mitochondrial fluorescence. Perinuclear aggregation of fluorescent mitochondria was observed when the human mitochondrial import receptor Tom20 (hTom20) was coexpressed with pOTC-GFP. Overexpression of hTom20 (not DeltahTom20, which lacks the anchor sequence) resulted in stimulated mitochondrial import of pOTC-GFP in COS-7 cells. When pOTC-GFP cDNA was microinjected into nuclei of human fibroblast cells, mitochondrial fluorescence was detected as early as 2-3 h after injection. These results show that GFP fusion protein can be used to visualize mitochondrial structures and to monitor mitochondrial protein import in a single cell in real time.