Kaplan R S, Mayor J A, Gremse D A, Wood D O
Department of Pharmacology, College of Medicine, University of South Alabama, Mobile 36688.
J Biol Chem. 1995 Feb 24;270(8):4108-14. doi: 10.1074/jbc.270.8.4108.
The gene encoding the mitochondrial citrate transport protein (CTP) in the yeast Saccharomyces cerevisiae has been identified, and its protein product has been overexpressed in Escherichia coli. The expressed CTP accumulates in inclusion bodies and can be solubilized with sarkosyl. Approximately 25 mg of solubilized CTP at a purity of 75% is obtained per liter of E. coli culture. The function of the solubilized CTP has been reconstituted in a liposomal system where both its kinetic parameters (i.e. Km = 0.36 mM and Vmax = 2.5 mumol/min/mg protein) and its substrate specificity have been determined. Notably, the yeast CTP displays a stricter specificity for tricarboxylates than do CTPs from higher eukaryotic organisms. Dot matrix analysis of the yeast CTP sequence indicates the presence of three homologous sequence domains (each approximately 100 residues in length), which are also related to domains in other CTPs. Thus, the yeast CTP displays the tripartite structure characteristic of other mitochondrial transporters. Alignment of the yeast CTP sequence with CTPs from other sources defines a consensus sequence that displays 89 positions of amino acid identity, as well as the more generalized mitochondrial transporter-associated sequence motif. Based on hydropathy analysis, the yeast CTP contains six putative membrane-spanning alpha-helices. Finally, Southern blot analysis indicates that the yeast genome contains a single gene encoding the mitochondrial CTP. Our data indicate that, based on both its structural and functional properties, the expressed yeast CTP can be assigned membership in the mitochondrial carrier family. The identification of the yeast CTP gene, and the expression and purification of large quantities of its protein product, pave the way for investigations into the roles of specific amino acids in the CTP translocation mechanism, as well as for the initiation of crystallization trials.
已鉴定出酿酒酵母中编码线粒体柠檬酸转运蛋白（CTP）的基因，其蛋白质产物已在大肠杆菌中过表达。表达的CTP积聚在包涵体中，可用十二烷基肌氨酸钠溶解。每升大肠杆菌培养物可获得约25毫克纯度为75%的溶解CTP。溶解的CTP的功能已在脂质体系统中重建，在该系统中已确定了其动力学参数（即Km = 0.36 mM和Vmax = 2.5 μmol/min/mg蛋白质）及其底物特异性。值得注意的是，酵母CTP对三羧酸的特异性比来自高等真核生物的CTP更严格。酵母CTP序列的点阵分析表明存在三个同源序列结构域（每个长度约100个残基），它们也与其他CTP中的结构域相关。因此，酵母CTP显示出其他线粒体转运蛋白的三方结构特征。酵母CTP序列与其他来源的CTP的比对定义了一个共有序列，该序列显示出89个氨基酸相同的位置，以及更普遍的线粒体转运蛋白相关序列基序。基于亲水性分析，酵母CTP包含六个假定的跨膜α螺旋。最后，Southern印迹分析表明酵母基因组包含一个编码线粒体CTP的单一基因。我们的数据表明，基于其结构和功能特性，表达的酵母CTP可被归入线粒体载体家族。酵母CTP基因的鉴定以及其大量蛋白质产物的表达和纯化，为研究特定氨基酸在CTP转运机制中的作用以及启动结晶试验铺平了道路。
