Probing the Role of Positive Residues in the ADP/ATP Carrier from Yeast. The Effect of Six Arginine Mutations on Oxidative Phosphorylation and AAC Expression
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- 作者：Veronika Mü ; ller ; Gabriele Basset ; David R. Nelson ; and Martin Klingenberg
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：December 17, 1996
- 年：1996
- 卷：35
- 期：50
- 页码：16132 - 16143
- 全文大小：912K
- 年卷期：v.35,no.50(December 17, 1996)
- ISSN：1520-4995
文摘
ADP/ATP transport is the terminal step of oxidativephosphorylation in mitochondria. In thispaper seven mutants of AAC2 from Saccharomyces cerevisiaeare studied on the cellular and mitochondriallevel. Six conspicuously located arginines were mutated intomostly neutral residues [Nelson, D. R.,Lawson, J. E., Klingenberg, M., & Douglas, M. G. (1993) J. Mol.Biol. 230, 1159-1170]. R96A, R96H,R204L, and R294A are located in the second transmembrane helix of eachrepeat while R252I, R253I,and R254I are in the arginine triplet of the last domain. All sixarginine residues are conserved in allknown ADP/ATP carrier sequences. At the cellular level, oxidativephosphorylation in R96H and R294Aretains 8% of the wild-type rate, but it is virtually zero in theother mutants. However, cytochrome c, aparameter of oxidative capacity, remains at 4-42% of wt. Theweak coordination of respiratory chainand AAC expression indicates that respiration is needed also for otherpurposes. In mitochondria theAAC-linked ATP synthesis is measured and segregated by using the AACinhibitor bongkrekate (BKA).Only the R96H and R294A mutants express a significant rate ofAAC-dependent ATP synthesis amountingto 2-18% of the plasmid-borne wild-type AAC2 mitochondria. Inall other mutants it is virtually zero.However, respiratory capacity and cytochrome c contentare reduced only by 20-70%. Whereas inimmunoblots the presence of AAC is detected in all mutant mitochondria,by quantitative ELISA noAAC can be measured down to 0.05 
r.gif">mol of AAC dimer/g of protein inR96A and R204L, whereas inR96H, R252I, R253I, and R254I the content is around 0.2 and in R294Athe content is 0.46 as comparedto 0.6 in the plasmid wild type. Also the[3H]CAT and [3H]BKA binding isvirtually zero in some mutantsand closely parallels the ELISA-determined AAC content, indicating thatthe mutations did not affect theinhibitor binding site. The turnover of AAC[V(ATP)/AAC content] in oxidative phosphorylation isreducedto 10% or 20% except for the two intrahelical mutants R96H and R294A.In the three Arg triplet mutants,it is nearly zero. In conclusion, the first two intrahelicalarginines R96 and R204, are essential for expressionbut probably also for the activity of AAC. R294A still retainsgood transport activity and a remarkablyhigh expression of AAC. All arginines in the triplet 252, 253, 254are essential. Extrapolation of the invitro phosphorylation rates to the cellular level bythe cytochrome c factor reveals a large discrepancytothe in vivo rates in particular for R294A. Thisindicates that these mutations render the AAC moresensitive to the regulatory intracellular ATP/ADP ratio than the wtAAC.
r.gif">mol of AAC dimer/g of protein inR96A and R204L, whereas inR96H, R252I, R253I, and R254I the content is around 0.2 and in R294Athe content is 0.46 as comparedto 0.6 in the plasmid wild type. Also the[3H]CAT and [3H]BKA binding isvirtually zero in some mutantsand closely parallels the ELISA-determined AAC content, indicating thatthe mutations did not affect theinhibitor binding site. The turnover of AAC[V(ATP)/AAC content] in oxidative phosphorylation isreducedto 10% or 20% except for the two intrahelical mutants R96H and R294A.In the three Arg triplet mutants,it is nearly zero. In conclusion, the first two intrahelicalarginines R96 and R204, are essential for expressionbut probably also for the activity of AAC. R294A still retainsgood transport activity and a remarkablyhigh expression of AAC. All arginines in the triplet 252, 253, 254are essential. Extrapolation of the invitro phosphorylation rates to the cellular level bythe cytochrome c factor reveals a large discrepancytothe in vivo rates in particular for R294A. Thisindicates that these mutations render the AAC moresensitive to the regulatory intracellular ATP/ADP ratio than the wtAAC.