Proteomics gives insight into the regulatory function of chloroplast thioredoxins
Proteomics gives insight into the regulatory function of chloroplast thioredoxins
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DOI：
10.1073/pnas.232703799
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发表时间：
2003-01-07
影响因子：
11.1
通讯作者：
Buchanan, BB
中科院分区：
文献类型：
--
作者：
Balmer, Y;Koller, A;Buchanan, BB
硫氧还蛋白是一种小的多功能氧化还原活性蛋白，广泛分布于生物体中。在叶绿体中，两种类型的硫氧还蛋白（f和m）共存，并在调节酶的活性中发挥核心作用。叶绿体中硫氧还蛋白的还原由铁-硫二硫化物酶（铁氧还蛋白-硫氧还蛋白还原酶）催化，铁氧还蛋白-硫氧还蛋白还原酶从铁氧还蛋白接收光合电子，从而提供光和酶活性之间的联系。叶绿体硫氧还蛋白在调节卡尔文循环和相关过程中起作用。然而，已知的硫氧还蛋白连接蛋白的数量相对较少（约16个），这增加了其他蛋白仍有待鉴定的可能性。为了追求这个机会，我们突变了硫氧还蛋白f和m，使得活性二硫化物的掩埋半胱氨酸被丝氨酸或丙氨酸取代，并将它们结合到亲和柱上以捕获叶绿体基质的靶蛋白。用DTT洗脱共价连接的蛋白质，在凝胶上分离，并通过质谱法鉴定。这种方法导致15个潜在的目标，功能在10个叶绿体过程中不知道是硫氧还蛋白连接的鉴定。包括似乎在质体到细胞核的信号传导和以前未被认识的氧化调节类型中起作用的蛋白质。这些靶标中约三分之二含有保守的半胱氨酸。我们还确定了11个以前未知的和9个确认的靶蛋白，这些蛋白是已知由硫氧还蛋白调控的途径的成员。与单个酶测定的结果相反，在亲和层析上没有观察到对硫氧还蛋白F或M的特异性。
Thioredoxins are small multifunctional redox active proteins widely if not universally distributed among living organisms. In chloroplasts, two types of thioredoxins (f and m) coexist and play central roles in regulating enzyme activity. Reduction of thioredoxins in chloroplasts is catalyzed by an iron-sulfur disulfide enzyme, ferredoxin-thioredoxin reductase, that receives photosynthetic electrons from ferredoxin, thereby providing a link between light and enzyme activity. Chloroplast thioredoxins function in the regulation of the Calvin cycle and associated processes. However, the relatively small number of known thioredoxin-linked proteins (about 16) raised the possibility that others remain to be identified. To pursue this opportunity, we have mutated thioredoxins f and m, such that the buried cysteine of the active disulfide has been replaced by serine or alanine, and bound them to affinity columns to trap target proteins of chloroplast stroma. The covalently linked proteins were eluted with DTT, separated on gels, and identified by mass spectrometry. This approach led to the identification of 15 potential targets that function in 10 chloroplast processes not known to be thioredoxin linked. Included are proteins that seem to function in plastid-to-nucleus signaling and in a previously unrecognized type of oxidative regulation. Approximately two-thirds of these targets contained conserved cysteines. We also identified 11 previously unknown and 9 confirmed target proteins that are members of pathways known to be regulated by thioredoxin. in contrast to results with individual enzyme assays, specificity for thioredoxin f or m was not observed on affinity chromatography.