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首页> 外文期刊>Biochemistry >Conformation-Dependent Swinging of the Matrix Loop m2 of the Mitochondrial Saccharomyces cerevisiae ADP/ATP Carrier
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Conformation-Dependent Swinging of the Matrix Loop m2 of the Mitochondrial Saccharomyces cerevisiae ADP/ATP Carrier

机译：形态依赖的线粒体酿酒酵母ADP / ATP载体的矩阵环m2的摆动

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Structure-function relationships of the membrane-embedded Saccharomyces cerevisiae mitochondrial ADP/ATP carrier were investigated through two independent approaches,namely,limited proteolysis and cysteine labeling.Experiments were carried out in the presence of either carboxyatractyloside(CATR)or bongkrekic acid(BA),two specific inhibitors of the ADP/ATP transport that bind to two distinct conformers involved in the translocation process.The proteolysis approach allowed us to demonstrate(i)that N-and C-terminal extremities of ADP/ATP carrier are facing the intermembrane space and(ii)that the central region of the carrier corresponding to the matrix loop m2 is accessible to externally added trypsin in a conformation-sensitive manner,being cleaved at the Lys163-Gly164 and Lys178-Thr179 bonds in the carrier-CATR and the carrier-BA complexes,respectively.The cysteine labeling approach was carried out on the S161C mutant of the ADP/ATP carrier.This variant of the carrier is fully active,displaying nucleotide transport kinetic parameters and inhibitor binding properties similar to that of wild-type carrier.Alkylation experiments,carried out on mitochondria with the nonpermeable reagents eosin-5-maleimide and iodoacetamidyl-3,6-dioxaoctanediamine-biotin,showed that Cys 161 is accessible from the outside in the carrier-CATR complex,whereas it is masked in the carrier-BA complex.Taken together,our results indicate that the matrix loop m2 connecting the transmembrane helices H3 to H4 intrudes to some extent into the inner mitochondrial membrane.Its participation in the translocation of ADP/ATP is strongly suggested,based on the finding that its accessibility to reagents added outside mitochondria is modified according to the conformational state of the carrier.
机译：通过有限的蛋白水解和半胱氨酸标记两种独立的方法研究了膜包埋的酿酒酵母线粒体ADP / ATP载体的结构-功能关系。在羧基白术苷（CATR）或邦克雷奇酸（BA）存在下进行了实验，两种特定的ADP / ATP转运抑制剂结合到转运过程中涉及的两个不同的构象异构体上。蛋白水解方法使我们能够证明（i）ADP / ATP载体的N和C末端都面向膜间空间。 （ii）外部添加的胰蛋白酶可以以构象敏感的方式接近对应于基质环m2的载体的中心区域，并在载体-CATR和载体中的Lys163-Gly164和Lys178-Thr179键处裂解-BA复合物。半胱氨酸标记方法是在ADP / ATP载体的S161C突变体上进行的。与野生型载体相似，其核苷酸转运动力学参数和抑制剂的结合特性相似。在线粒体上用不可渗透试剂曙红-5-马来酰亚胺和碘乙酰胺基-3,6-二氧八辛二胺-生物素进行烷基化实验，表明Cys 161是综合起来，我们的结果表明，连接跨膜螺旋H3至H4的基质环m2在某种程度上侵入线粒体内膜。强烈建议其参与ADP / ATP的转运，这是基于其对线粒体外部添加的试剂的可及性随载体的构象状态而改变的发现。

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