The delivery of ADP/ATP carrier protein to mitochondria probed by fusions with green fluorescent protein and β-galactosidase

• Published in: FEMS yeast research Vol. 4; no. 3; pp. 315 - 321
• Main Authors: Polčicová, Katarı́na, Kempná, Petra, Šabová, L’udmila, Gavurnı́ková, Gabriela, Polčic, Peter, Kolarov, Jordan
• Format: Journal Article
• Language: English
• Published: Oxford, UK Elsevier B.V 01-12-2003; Blackwell Publishing Ltd; Oxford University Press
• Subjects: AAC, ADP/ATP carrier; ADP/ATP carrier; ADP/ATP carrier protein; beta-Galactosidase - genetics; beta-Galactosidase - metabolism; Biogenesis; DAPI, 4′,6′‐diamidino‐2‐phenyl‐indole; Genes; Genes, Fungal; Genes, Reporter; GFP, green fluorescent protein; Green fluorescent protein; Green Fluorescent Proteins; lacZ, β‐galactosidase; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Mitochondria; Mitochondria - enzymology; Mitochondrial ADP, ATP Translocases - genetics; Mitochondrial ADP, ATP Translocases - metabolism; Mitochondrion; Protein import; Protein transport; Proteinase; Proteins; Recombinant Fusion Proteins - metabolism; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; SM, synthetic medium; Tim, translocase of inner mitochondrial membrane; Tom, translocase of outer mitochondrial membrane; β-Galactosidase; Mitochondrion; Tim, translocase of inner mitochondrial membrane; GFP, green fluorescent protein; DAPI, 4′,6′-diamidino-2-phenyl-indole; lacZ, β-galactosidase; Tom, translocase of outer mitochondrial membrane; Protein import; SM, synthetic medium; Biogenesis; AAC, ADP/ATP carrier; ADP/ATP carrier
• ISSN: 1567-1356; 1567-1364
• DOI: 10.1016/S1567-1356(03)00170-3

The import of proteins into mitochondria is an essential process, largely investigated in vitro with isolated mitochondria and radioactively labeled precursors. In this study, we used intact cells and fusions with genes encoding two reporter proteins, green fluorescent protein (GFP) and β-galactosidase (
lacZ), to probe the import of the ADP/ATP carrier (AAC). Typical mitochondrial fluorescence was observed with AAC-GFP fusions containing at least one complete transmembrane loop. This confirms the results of in vitro analysis demonstrating that an internal targeting signal was present in each one of the three transmembrane loops of the carrier. The fusions of AAC fragments to β-galactosidase demonstrated that the targeting signal was capable of delivering the reporter molecule to the mitochondrial surface, but not to internalize it to a protease-inaccessible location. The delivery to a protease-inaccessible location required the presence of more distal sequences present within the third (C-terminal) transmembrane loop of the carrier molecule. The results of our study provide an alternative for investigation in a natural context of mitochondrial protein import in cells when the isolation of intact, functional mitochondria is not achievable.