Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA.
Plant Cell. 2011 Apr;23(4):1273-92. doi: 10.1105/tpc.111.084400. Epub 2011 Apr 15.
In this work, we query the Chlamydomonas reinhardtii copper regulon at a whole-genome level. Our RNA-Seq data simulation and analysis pipeline validated a 2-fold cutoff and 10 RPKM (reads per kilobase of mappable length per million mapped reads) (~1 mRNA per cell) to reveal 63 CRR1 targets plus another 86 copper-responsive genes. Proteomic and immunoblot analyses captured 25% of the corresponding proteins, whose abundance was also dependent on copper nutrition, validating transcriptional regulation as a major control mechanism for copper signaling in Chlamydomonas. The impact of copper deficiency on the expression of several O₂-dependent enzymes included steps in lipid modification pathways. Quantitative lipid profiles indicated increased polyunsaturation of fatty acids on thylakoid membrane digalactosyldiglycerides, indicating a global impact of copper deficiency on the photosynthetic apparatus. Discovery of a putative plastid copper chaperone and a membrane protease in the thylakoid suggest a mechanism for blocking copper utilization in the chloroplast. We also found an example of copper sparing in the N assimilation pathway: the replacement of copper amine oxidase by a flavin-dependent backup enzyme. Forty percent of the targets are previously uncharacterized proteins, indicating considerable potential for new discovery in the biology of copper.
在这项工作中，我们在全基因组水平上对莱茵衣藻的铜调控组进行了查询。我们的 RNA-Seq 数据模拟和分析管道验证了 2 倍的截止值和 10 RPKM（每百万映射读取中每千碱基映射长度的读取数）（~1 个 mRNA 每个细胞），以揭示 63 个 CRR1 靶标加上另外 86 个铜反应基因。蛋白质组学和免疫印迹分析捕获了相应蛋白质的 25%，其丰度也依赖于铜营养，这验证了转录调控是铜信号在衣藻中的主要控制机制。铜缺乏对几种 O₂依赖酶的表达的影响包括脂质修饰途径的步骤。定量脂质谱表明，类囊体膜二半乳糖基二甘油的脂肪酸的多不饱和性增加，表明铜缺乏对光合作用装置的全局影响。在类囊体中发现了一种假定的质体铜伴侣和一种膜蛋白酶，这表明了一种阻止叶绿体中铜利用的机制。我们还在氮同化途径中发现了一个铜节约的例子：铜胺氧化酶被黄素依赖性备用酶取代。40%的靶标是以前未被表征的蛋白质，这表明在铜生物学中有很大的新发现潜力。
