Müller V, Basset G, Nelson D R, Klingenberg M
Institute for Physical Biochemistry, University of Munich, Germany.
Biochemistry. 1996 Dec 17;35(50):16132-43. doi: 10.1021/bi960667r.
ADP/ATP transport is the terminal step of oxidative phosphorylation in mitochondria. In this paper seven mutants of AAC2 from Saccharomyces cerevisiae are studied on the cellular and mitochondrial level. Six conspicuously located arginines were mutated into mostly 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 each repeat while R252I, R253I, and R254I are in the arginine triplet of the last domain. All six arginine residues are conserved in all known ADP/ATP carrier sequences. At the cellular level, oxidative phosphorylation in R96H and R294A retains 8% of the wild-type rate, but it is virtually zero in the other mutants. However, cytochrome c, a parameter of oxidative capacity, remains at 4-42% of wt. The weak coordination of respiratory chain and AAC expression indicates that respiration is needed also for other purposes. In mitochondria the AAC-linked ATP synthesis is measured and segregated by using the AAC inhibitor bongkrekate (BKA). Only the R96H and R294A mutants express a significant rate of AAC-dependent ATP synthesis amounting to 2-18% of the plasmid-borne wild-type AAC2 mitochondria. In all other mutants it is virtually zero. However, respiratory capacity and cytochrome c content are reduced only by 20-70%. Whereas in immunoblots the presence of AAC is detected in all mutant mitochondria, by quantitative ELISA no AAC can be measured down to 0.05 mumol of AAC dimer/g of protein in R96A and R204L, whereas in R96H, R2521, R2531, and R254I the content is around 0.2 and in R294A the content is 0.46 as compared to 0.6 in the plasmid wild type. Also the [3H]CAT and [3H]BKA binding is virtually zero in some mutants and closely parallels the ELISA-determined AAC content, indicating that the mutations did not affect the inhibitor binding site. The turnover of AAC [V(ATP)/AAC content] in oxidative phosphorylation is reduced to 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 intrahelical arginines R96 and R204, are essential for expression but probably also for the activity of AAC. R294A still retains good transport activity and remarkably high expression of AAC. All arginines in the triplet 252, 253, 254 are essential. Extrapolation of the in vitro phosphorylation rates to the cellular level by the cytochrome c factor reveals a large discrepancy to the in vivo rates in particular for R294A. This indicates that these mutations render the AAC more sensitive to the regulatory intracellular ATP/ADP ratio than the wt AAC.
ADP/ATP转运是线粒体氧化磷酸化的最后一步。本文在细胞和线粒体水平上研究了酿酒酵母中七个AAC2突变体。六个位置显著的精氨酸被突变成大多为中性的残基[尼尔森，D.R.，劳森，J.E.，克林根贝格，M.，&道格拉斯，M.G.（1993）《分子生物学杂志》230，1159 - 1170]。R96A、R96H、R204L和R294A位于每个重复序列的第二个跨膜螺旋中，而R252I、R253I和R254I位于最后一个结构域的精氨酸三联体中。所有六个精氨酸残基在所有已知的ADP/ATP载体序列中都是保守的。在细胞水平上，R96H和R294A中的氧化磷酸化保留了野生型速率的8%，但在其他突变体中几乎为零。然而，作为氧化能力参数的细胞色素c仍保持在野生型的4 - 42%。呼吸链与AAC表达的弱协调性表明呼吸作用也用于其他目的。在线粒体中，通过使用AAC抑制剂邦克雷酸（BKA）来测量和区分与AAC相关的ATP合成。只有R96H和R294A突变体表现出显著的依赖AAC的ATP合成速率，达到质粒携带的野生型AAC2线粒体的2 - 18%。在所有其他突变体中，该速率几乎为零。然而，呼吸能力和细胞色素c含量仅降低了20 - 70%。在免疫印迹中，在所有突变体线粒体中都检测到了AAC的存在，但通过定量ELISA，在R96A和R204L中，低至0.05 μmol AAC二聚体/克蛋白质时都无法检测到AAC，而在R96H、R252I、R253I和R254I中，含量约为0.2，在R294A中含量为0.46，相比之下质粒野生型为0.6。同样，在一些突变体中，[³H]CAT和[³H]BKA结合几乎为零，并且与ELISA测定的AAC含量密切平行，表示这些突变没有影响抑制剂结合位点。除了两个螺旋内突变体R96H和R294A外，氧化磷酸化中AAC的周转率[V(ATP)/AAC含量]降低到10%或20%。在三个精氨酸三联体突变体中，周转率几乎为零。总之，前两个螺旋内精氨酸R96和R204对AAC的表达至关重要，可能对其活性也至关重要。R294A仍保留良好的转运活性和显著高的AAC表达。三联体252、253、254中的所有精氨酸都是必不可少的。通过细胞色素c因子将体外磷酸化速率外推到细胞水平，发现与体内速率存在很大差异，特别是对于R294A。这表明这些突变使AAC比野生型AAC对细胞内ATP/ADP比率的调节更敏感。
