Department of Plant Biology, Cornell University, Ithaca, New York 14853, USA.
Mol Cell Proteomics. 2010 Jul;9(7):1594-615. doi: 10.1074/mcp.M000038-MCP201. Epub 2010 Apr 26.
To characterize MDa-sized macromolecular chloroplast stroma protein assemblies and to extend coverage of the chloroplast stroma proteome, we fractionated soluble chloroplast stroma in the non-denatured state by size exclusion chromatography with a size separation range up to approximately 5 MDa. To maximize protein complex stability and resolution of megadalton complexes, ionic strength and composition were optimized. Subsequent high accuracy tandem mass spectrometry analysis (LTQ-Orbitrap) identified 1081 proteins across the complete native mass range. Protein complexes and assembly states above 0.8 MDa were resolved using hierarchical clustering, and protein heat maps were generated from normalized protein spectral counts for each of the size exclusion chromatography fractions; this complemented previous analysis of stromal complexes up to 0.8 MDa (Peltier, J. B., Cai, Y., Sun, Q., Zabrouskov, V., Giacomelli, L., Rudella, A., Ytterberg, A. J., Rutschow, H., and van Wijk, K. J. (2006) The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts. Mol. Cell. Proteomics 5, 114-133). This combined experimental and bioinformatics analyses resolved chloroplast ribosomes in different assembly and functional states (e.g. 30, 50, and 70 S), which enabled the identification of plastid homologues of prokaryotic ribosome assembly factors as well as proteins involved in co-translational modifications, targeting, and folding. The roles of these ribosome-associating proteins will be discussed. Known RNA splice factors (e.g. CAF1/WTF1/RNC1) as well as uncharacterized proteins with RNA-binding domains (pentatricopeptide repeat, RNA recognition motif, and chloroplast ribosome maturation), RNases, and DEAD box helicases were found in various sized complexes. Chloroplast DNA (>3 MDa) was found in association with the complete heteromeric plastid-encoded DNA polymerase complex, and a dozen other DNA-binding proteins, e.g. DNA gyrase, topoisomerase, and various DNA repair enzymes. The heteromeric >or=5-MDa pyruvate dehydrogenase complex and the 0.8-1-MDa acetyl-CoA carboxylase complex associated with uncharacterized biotin carboxyl carrier domain proteins constitute the entry point to fatty acid metabolism in leaves; we suggest that their large size relates to the need for metabolic channeling. Protein annotations and identification data are available through the Plant Proteomics Database, and mass spectrometry data are available through Proteomics Identifications database.
为了描绘 MDa 大小的叶绿体基质大分子蛋白质组装体，并扩展叶绿体基质蛋白质组的覆盖范围，我们通过大小排阻色谱法在非变性状态下对可溶性叶绿体基质进行了分级，大小分离范围高达约 5 MDa。为了最大限度地提高蛋白质复合物的稳定性和解析兆道尔顿复合物的分辨率，优化了离子强度和组成。随后，通过高精度串联质谱分析（LTQ-Orbitrap）在整个天然质量范围内鉴定了 1081 种蛋白质。使用层次聚类解析了大于 0.8 MDa 的蛋白质复合物和组装状态，并为每个大小排阻色谱馏分生成了归一化蛋白质光谱计数的蛋白质热图；这补充了以前对 0.8 MDa 以下基质复合物的分析（Peltier，JB，Cai，Y.，Sun，Q.，Zabrouskov，V.，Giacomelli，L.，Rudella，A.，Ytterberg，AJ.，Rutschow，H.，和 van Wijk，KJ.（2006）拟南芥叶绿体基质的低聚体蛋白质组。Mol. Cell. Proteomics 5, 114-133）。这种结合的实验和生物信息学分析解决了不同组装和功能状态的叶绿体核糖体（例如 30、50 和 70 S），从而能够鉴定原核核糖体组装因子以及参与共翻译修饰、靶向和折叠的质体同源物。将讨论这些核糖体相关蛋白的作用。在各种大小的复合物中发现了已知的 RNA 剪接因子（例如 CAF1/WTF1/RNC1）以及具有 RNA 结合结构域（五肽重复，RNA 识别基序和叶绿体核糖体成熟），RNases 和 DEAD 盒解旋酶的未表征蛋白。在与完整的异源质体编码 DNA 聚合酶复合物以及其他十几个 DNA 结合蛋白（例如 DNA 回旋酶，拓扑异构酶和各种 DNA 修复酶）相关联的情况下发现了叶绿体 DNA（>3 MDa）。异源> = 5-MDa 丙酮酸脱氢酶复合物和 0.8-1-MDa 乙酰辅酶 A 羧化酶复合物与未表征的生物素羧基载体结构域蛋白相关，构成了叶片中脂肪酸代谢的切入点；我们认为它们的大尺寸与代谢通道化的需要有关。通过植物蛋白质组学数据库可获得蛋白质注释和鉴定数据，通过蛋白质组学鉴定数据库可获得质谱数据。
