RESUMO
cDNAs for green fluorescent protein (GFP) and for a GFP fusion protein containing the presequence of human ornithine transcarbamylase (pOTC-GFP) were transfected into cultured human fibroblasts. GFP cDNA gave diffuse fluorescence throughout the cytoplasm and the nucleus, whereas pOTC-GFP cDNA gave mitochondria-associated fluorescence. Fluorescent mitochondrial structures could be classified into five patterns: thread-like mitochondria, fine thread-like ones, rod-like ones, granular ones, and granular ones with weak cytosolic fluorescence. pOTC-GFP mutants resulted in a loss of mitochondrial fluorescence and an appearance of weak fluorescence throughout the cytoplasm. pOTC-GFP cDNA was transfected into fibroblasts from patients with various mitochondrial diseases. Higher ratios of fibroblasts with granular mitochondria and those with fine thread-like ones were observed in a patient with Reye's syndrome and a patient with Kearns-Sayre syndrome. Weak cytosolic fluorescence was sometimes observed in fibroblasts from these patients. This method will be useful to analyze mitochondrial structural alterations and disorders of mitochondrial protein import.
Assuntos
Proteínas Luminescentes/análise , Erros Inatos do Metabolismo/patologia , Mitocôndrias/patologia , Animais , Células Cultivadas , DNA Complementar/genética , Fibroblastos/química , Fibroblastos/classificação , Fibroblastos/patologia , Proteínas de Fluorescência Verde , Humanos , Síndrome de Kearns-Sayre/enzimologia , Síndrome de Kearns-Sayre/genética , Síndrome de Kearns-Sayre/patologia , Proteínas Luminescentes/genética , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/metabolismo , Microscopia de Fluorescência , Mitocôndrias/enzimologia , Mitocôndrias/genética , Ornitina Carbamoiltransferase/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Síndrome de Reye/enzimologia , Síndrome de Reye/genética , Síndrome de Reye/patologia , CifozoáriosRESUMO
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.
