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The yeast mitochondrial ADP/ATP carrier functions as a monomer in mitochondrial membranes.

ABSTRACT: Mitochondrial carriers are believed widely to be dimers both in structure and function. However, the structural fold is a barrel of six transmembrane alpha-helices without an obvious dimerisation interface. Here, we show by negative dominance studies that the yeast mitochondrial ADP/ATP carrier 2 from Saccharomyces cerevisiae (AAC2) is functional as a monomer in the mitochondrial membrane. Adenine nucleotide transport by wild-type AAC2 is inhibited by the sulfhydryl reagent 2-sulfonatoethyl-methanethiosulfonate (MTSES), whereas the activity of a mutant AAC2, devoid of cysteines, is unaffected. Wild-type and cysteine-less AAC2 were coexpressed in different molar ratios in yeast mitochondrial membranes. After addition of MTSES the residual transport activity correlated linearly with the fraction of cysteine-less carrier present in the membranes, and so the two versions functioned independently of each other. Also, the cysteine-less and wild-type carriers were purified separately, mixed in defined ratios and reconstituted into liposomes. Again, the residual transport activity in the presence of MTSES depended linearly on the amount of cysteine-less carrier. Thus, the entire transport cycle for ADP/ATP exchange is carried out by the monomer.

SUBMITTER: Bamber L

PROVIDER: S-EPMC1891095 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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The yeast mitochondrial ADP/ATP carrier functions as a monomer in mitochondrial membranes.

Bamber Lisa L Harding Marilyn M Monné Magnus M Slotboom Dirk-Jan DJ Kunji Edmund R S ER

Proceedings of the National Academy of Sciences of the United States of America 20070612 26

Mitochondrial carriers are believed widely to be dimers both in structure and function. However, the structural fold is a barrel of six transmembrane alpha-helices without an obvious dimerisation interface. Here, we show by negative dominance studies that the yeast mitochondrial ADP/ATP carrier 2 from Saccharomyces cerevisiae (AAC2) is functional as a monomer in the mitochondrial membrane. Adenine nucleotide transport by wild-type AAC2 is inhibited by the sulfhydryl reagent 2-sulfonatoethyl-meth ...[more]

PMID: 17566106

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Project description:An increasing number of commonly prescribed drugs are known to interfere with mitochondrial function, causing cellular toxicity, but the underlying mechanisms are largely unknown. Although often not considered, mitochondrial transport proteins form a significant class of potential mitochondrial off-targets. So far, most drug interactions have been reported for the mitochondrial ADP/ATP carrier (AAC), which exchanges cytosolic ADP for mitochondrial ATP. Here, we show inhibition of cellular respiratory capacity by only a subset of the 18 published AAC inhibitors, which questions whether all compound do indeed inhibit such a central metabolic process. This could be explained by the lack of a simple, direct model system to evaluate and compare drug-induced AAC inhibition. Methods: For its development, we have expressed and purified human AAC1 (hAAC1) and applied two approaches. In the first, thermostability shift assays were carried out to investigate the binding of these compounds to human AAC1. In the second, the effect of these compounds on transport was assessed in proteoliposomes with reconstituted human AAC1, enabling characterization of their inhibition kinetics. Results: Of the proposed inhibitors, chebulinic acid, CD-437 and suramin are the most potent with IC50-values in the low micromolar range, whereas another six are effective at a concentration of 100 μM. Remarkably, half of all previously published AAC inhibitors do not show significant inhibition in our assays, indicating that they are false positives. Finally, we show that inhibitor strength correlates with a negatively charged surface area of the inhibitor, matching the positively charged surface of the substrate binding site. Conclusion: Consequently, we have provided a straightforward model system to investigate AAC inhibition and have gained new insights into the chemical compound features important for inhibition. Better evaluation methods of drug-induced inhibition of mitochondrial transport proteins will contribute to the development of drugs with an enhanced safety profile.

Dataset Information

The yeast mitochondrial ADP/ATP carrier functions as a monomer in mitochondrial membranes.

Publications

The yeast mitochondrial ADP/ATP carrier functions as a monomer in mitochondrial membranes.

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