Molecular Identification and Functional Characterization of Arabidopsis thaliana Mitochondrial and Chloroplastic NAD+ Carrier Proteins
Molecular Identification and Functional Characterization of Arabidopsis thaliana Mitochondrial and Chloroplastic NAD+ Carrier Proteins
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DOI：
10.1074/jbc.m109.041830
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发表时间：
2009-11-06
影响因子：
4.8
通讯作者：
Neuhaus, H. Ekkehard
中科院分区：
文献类型：
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作者：
Palmieri, Ferdinando;Rieder, Benjamin;Neuhaus, H. Ekkehard
拟南芥（Arabidopsis thaliana L.）基因组包含58个属于线粒体载体家族的膜蛋白。两个线粒体载体家族成员（此处命名为AtNDT 1和AtNDT 2）与来自面包酵母的线粒体烟酰胺腺嘌呤二核苷酸（NAD（+））载体ScNDT 1表现出高度的结构相似性。AtNDT 1或AtNDT 2的表达恢复了缺乏ScNDT的酵母突变体中线粒体NAD（+）转运活性。利用绿色荧光蛋白融合蛋白进行的定位研究表明，AtNDT 1定位于叶绿体，而AtNDT 2定位于线粒体。在大肠杆菌中进行异源表达，然后进行纯化、在蛋白脂质体中重构和摄取实验，结果表明，两种载体对NAD（+）都表现出亚毫摩尔亲和力，并以反交换模式转运该化合物。在各种底物中，ADP和AMP是NAD（+）最有效的反交换底物。AtB 1-和AtB 2-启动子-GUS植物表明，这两种基因在发育中的组织中，特别是在高代谢活性细胞中强烈表达。这两种载体的存在下讨论相对于在植物中从头NAD（+）生物合成的亚细胞定位和相对于NAD（+）依赖的代谢途径和叶绿体和线粒体的氧化还原平衡。
The Arabidopsis thaliana L. genome contains 58 membrane proteins belonging to the mitochondrial carrier family. Two mitochondrial carrier family members, here named AtNDT1 and AtNDT2, exhibit high structural similarities to the mitochondrial nicotinamide adenine dinucleotide (NAD(+)) carrier ScNDT1 from bakers' yeast. Expression of AtNDT1 or AtNDT2 restores mitochondrial NAD(+) transport activity in a yeast mutant lacking ScNDT. Localization studies with green fluorescent protein fusion proteins provided evidence that AtNDT1 resides in chloroplasts, whereas only AtNDT2 locates to mitochondria. Heterologous expression in Escherichia coli followed by purification, reconstitution in proteoliposomes, and uptake experiments revealed that both carriers exhibit a submillimolar affinity for NAD(+) and transport this compound in a counterexchange mode. Among various substrates ADP and AMP are the most efficient counter-exchange substrates for NAD(+). Atndt1- and Atndt2-promoter-GUS plants demonstrate that both genes are strongly expressed in developing tissues and in particular in highly metabolically active cells. The presence of both carriers is discussed with respect to the subcellular localization of de novo NAD(+) biosynthesis in plants and with respect to both the NAD(+)-dependent metabolic pathways and the redox balance of chloroplasts and mitochondria.