Formation of a cytoplasmic salt bridge network in the matrix state is a fundamental step in the transport mechanism of the mitochondrial ADP/ATP carrier.
Formation of a cytoplasmic salt bridge network in the matrix state is a fundamental step in the transport mechanism of the mitochondrial ADP/ATP carrier.
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DOI：
10.1016/j.bbabio.2015.09.013
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发表时间：
2016-01
期刊：
影响因子：
--
通讯作者：
Kunji ERS
中科院分区：
文献类型：
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作者：
King MS;Kerr M;Crichton PG;Springett R;Kunji ERS
线粒体ADP/ATP载体催化ADP和ATP跨线粒体内膜的等摩尔交换。在结构上，它们由具有单个底物结合位点的三个同源结构域组成。它们在细胞质和基质状态之间交替，其中结合位点可接近这些隔室以结合ADP或ATP。有人提出，状态之间的循环发生的破坏和形成的矩阵和细胞质盐桥网络在一个交替的方式，但后者的形成还没有被实验证明。在这里，我们表明，状态依赖性的细胞质盐桥网络的形成，可以证明通过测量的影响，突变的热稳定性的洗涤剂溶解载体锁定在一个特定的状态。为此，进行突变以增加或减少细胞质网络的总体相互作用能。当锁定在细胞质状态的抑制剂carboxyloxylside，突变体和野生型载体的热稳定性是相似的，但当锁定在矩阵状态的抑制剂bongkrekic酸，它们与预测的相互作用能的细胞质网络，证明其形成。改变细胞质网络的相互作用能也对运输动力学产生了深远的影响，表明网络的形成是运输循环中的关键步骤。这些结果是一致的一个独特的交替访问机制，涉及同时旋转的三个域周围的中央易位途径。线粒体ADP/ATP载体在基质和细胞质状态之间交替。基质和细胞质盐桥网络调节中央结合位点的进入。热稳定性测定用于探测载体中的状态依赖性相互作用。细胞质盐桥网络突变只影响基质状态的热稳定性。细胞质网络的形成是运输循环中的基本步骤。
Mitochondrial ADP/ATP carriers catalyze the equimolar exchange of ADP and ATP across the mitochondrial inner membrane. Structurally, they consist of three homologous domains with a single substrate binding site. They alternate between a cytoplasmic and matrix state in which the binding site is accessible to these compartments for binding of ADP or ATP. It has been proposed that cycling between states occurs by disruption and formation of a matrix and cytoplasmic salt bridge network in an alternating way, but formation of the latter has not been shown experimentally. Here, we show that state-dependent formation of the cytoplasmic salt bridge network can be demonstrated by measuring the effect of mutations on the thermal stability of detergent-solubilized carriers locked in a specific state. For this purpose, mutations were made to increase or decrease the overall interaction energy of the cytoplasmic network. When locked in the cytoplasmic state by the inhibitor carboxyatractyloside, the thermostabilities of the mutant and wild-type carriers were similar, but when locked in the matrix state by the inhibitor bongkrekic acid, they correlated with the predicted interaction energy of the cytoplasmic network, demonstrating its formation. Changing the interaction energy of the cytoplasmic network also had a profound effect on the kinetics of transport, indicating that formation of the network is a key step in the transport cycle. These results are consistent with a unique alternating access mechanism that involves the simultaneous rotation of the three domains around a central translocation pathway. Mitochondrial ADP/ATP carriers alternate between the matrix and cytoplasmic state. Matrix and cytoplasmic salt bridge networks regulate access to central binding site. Thermostability assays are used to probe state-dependent interactions in carriers. Cytoplasmic salt bridge network mutations only affect matrix state thermostability. Formation of the cytoplasmic network is a fundamental step in the transport cycle.