The dynamic dimerization of the yeast ADP/ATP carrier in the inner mitochondrial membrane is affected by conserved cysteine residues.
The ADP/ATP carrier (AAC) that facilitates the translocation of ATP made in mitochondria is inserted at the inner mitochondrial membrane by the TIM10-TIM22 protein import system. Here we addressed the state of the AAC precursor during insertion (stage IV of import) and identified residues of the carrier important for dimerization. By a combination of (i) import of a mix of His-tagged and untagged versions of AAC either 35S-labeled or unlabeled, (ii) import of a tandem covalent dimer AAC into wild-type mitochondria, and (iii) import of monomeric AAC into mitochondria expressing only the tandem covalent dimer AAC, we found that the stage IV intermediate is a monomer, and this stage is probably the rate-limiting step of insertion in the membrane. Subsequent dimerization occurs extremely rapidly (within less than a minute). The incoming monomer dimerizes with monomeric endogenous AAC suggesting that the AAC dimer is very dynamic. Conserved Cys residues were found not to affect insertion significantly, but they are crucial for the dimerization process to obtain a functional carrier.
Dyall
Sabrina D
SD
School of Biological Sciences, 2.205 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
Agius
Stephanie C
SC
De Marcos Lousa
Carine
C
Trezeguet
Veronique
V
Tokatlidis
Kostas
K
D001483:Base Sequence; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry; D004271:DNA, Fungal / Q000235:genetics; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D007425:Intracellular Membranes / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D000226:Mitochondrial ADP, ATP Translocases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004271:DNA, Fungal; D004220:Disulfides; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D000226:Mitochondrial ADP, ATP Translocases; D003545:Cysteine