Oligomeric state of wild-type and cysteine-less yeast mitochondrial citrate transport proteins.
Oligomeric state of wild-type and cysteine-less yeast mitochondrial citrate transport proteins.
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野生型和无半胱氨酸酵母线粒体柠檬酸转运蛋白的寡聚状态。
DOI：
10.1023/a:1005460810527
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发表时间：
1999
影响因子：
3
通讯作者：
Kaplan,RS
中科院分区：
文献类型：
--
作者：
Kotaria,R;Mayor,JA;Walters,DE;Kaplan,RS
对酿酒酵母（Saccharomycescerevisiae）线粒体柠檬酸转运蛋白（CTP）的寡聚状态进行了研究。野生型和无半胱氨酸CTP在E.用肌氨酸溶解。溶解的材料的纯度约为75%。当掺入磷脂囊泡中时，野生型和无Cys的CTP都获得了高的比转运活性，从而证明了制剂的结构和功能完整性。采用两种独立的方法测定天然分子量。首先，CTP分子量通过非变性尺寸排阻色谱法测定。用这种方法我们得到野生型和无Cys CTP的分子量分别为70，961和70，118。第二种方法是利用低浓度的带负电荷的洗涤剂sarkosyl进行电荷位移天然凝胶电泳，这既可以使CTP和蛋白标准品发生电荷位移，也可以提高蛋白的溶解度，通过第二种方法，我们获得了野生型和无Cys CTP的分子量值分别为69，122和74，911。这两种方法都清楚地表明，在溶解后，野生型和无Cys的CTP仅以二聚体存在。此外，二硫键不是二聚体形成或稳定所必需的。CTP的二聚体状态对于CTP易位机制的结构基础具有重要意义。
Experiments have been conducted to determine the oligomeric state of the mitochondrial citratetransport protein (CTP) from the yeastSaccharomyces cerevisiae. Both wild-type andcysteine-less (Cys-less) CTPs were overexpressed inE. coliand solubilized with sarkosyl. The purity ofthe solubilized material is approximately 75%. Upon incorporation into phospholipid vesicles, ahigh specific transport activity is obtained with both the wild-type and Cys-less CTPs, therebydemonstrating the structural and functional integrity of the preparations. Two independentapproaches were utilized to determine native molecular weight. First, CTP molecular weightwas determined via nondenaturing size-exclusion chromatography. With this methodology weobtained molecular weight values of 70,961 and 70,118 for the wild-type and Cys-less CTPs,respectively. Second, charge-shift native gel electrophoresis was carried out utilizing a lowconcentration of the negatively charged detergent sarkosyl, which served to both impart acharge shift to the CTP and the protein standards, as well as to promote protein solubility.Via the second method, we obtained molecular weight values of 69,122 and 74,911 forthe wild-type and Cys-less CTPs, respectively. Both methods clearly indicate that followingsolubilization, the wild-type and the Cys-less CTPs exist exclusively as dimers. Furthermore,disulfide bonds are not required for either dimer formation or stabilization. The dimericstate of the CTP has important implications for the structural basis underlying the CTPtranslocation mechanism.