title: 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.
authors.affiliation: Department of Molecular Genetics, Kumamoto University School of Medicine, Kumamoto 862, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Yano
authors.forename: M
authors.initials: M
authors.lastname: Kanazawa
authors.forename: K
authors.initials: K
authors.lastname: Terada
authors.forename: C
authors.initials: C
authors.lastname: Namchai
authors.forename: M
authors.initials: M
authors.lastname: Yamaizumi
authors.forename: B
authors.initials: B
authors.lastname: Hanson
authors.forename: N
authors.initials: N
authors.lastname: Hoogenraad
authors.forename: M
authors.initials: M
authors.lastname: Mori
mesh_terms: D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000737:chemistry; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008845:Microinjections; D008928:Mitochondria / Q000737:chemistry*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D016297:Mutagenesis, Site-Directed; D009954:Ornithine Carbamoyltransferase / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D008164:Luminescent Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D011498:Protein Precursors; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C084008:TOMM20 protein, human; D049452:Green Fluorescent Proteins; D009954:Ornithine Carbamoyltransferase