Site-directed Mutagenesis of the Yeast Mitochondrial ADP/ATP Translocator: Six Arginines and One Lysine are Essential

• Published in: Journal of molecular biology Vol. 230; no. 4; pp. 1159 - 1170
• Main Authors: Nelson, David R., Lawson, Janet E., Klingenberg, Martin, Douglas, Michael G.
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 20.04.1993; Elsevier
• Subjects: AAC2; aac2 protein; active transport; adenosine diphosphate; adenosine triphosphate; ADP/ATP translocator; Amino Acid Sequence; amino acid sequences; Analytical, structural and metabolic biochemistry; arginine; Base Sequence; Binding and carrier proteins; binding proteins; Biological and medical sciences; Carrier Proteins - classification; Carrier Proteins - genetics; Cell Fractionation; cell membranes; DNA Mutational Analysis; Fundamental and applied biological sciences. Psychology; Fungal Proteins - classification; Fungal Proteins - genetics; Gene Deletion; inner mitochondrial membranes; lysine; membrane proteins; mitochondria; Mitochondria - physiology; Mitochondrial ADP, ATP Translocases - genetics; Models, Molecular; Molecular Sequence Data; mutagenesis; Mutagenesis, Site-Directed; mutants; protein engineering; Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Sequence Analysis, DNA; Sequence Homology, Amino Acid; site-directed mutagenesis; structural genes; Structure-Activity Relationship; yeast; site-directed mutagenesis; AAC2; protein engineering; yeast; ADP/ATP translocator; Yeast; Site directed mutagenesis; ADP; In vitro; Structure function relationship; Internal membrane; Fungi; Mitochondria; Ascomycetes; ATP; Saccharomyces cerevisiae; Carrier protein; Thallophyta
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.1993.1233

The ADP/ATP translocator mediates adenine nucleotide exchange across the inner mitochondrial membrane. ADP/ATP exchange is essential when yeast are grown on a non-fermentable carbon source such as glycerol, but it is not required for growth on glucose. Failure to grow on glycerol is therefore a phenotypic indicator of protein function, and it has been used here to screen site-directed mutants to identify functionally important amino acids in the yeast adenine nucleotide translocator (AAC2). Single mutations of all four charged amino acids in the transmembrane segments of AAC2 (K38A, R96D, R96H, R96L, R96P, R204L, R294A) resulted in loss of function, as did mutations in the matrix arginine cluster (R252I, R253I, R254I). Seven other residues were mutated without affecting growth on glycerol (C73S, C244S, C271S, K179M, K182I, P247G, W235F). The non-functional mutants have been used to select intragenic suppressors to gain further insight into the structure of this membrane transport protein.