Firmino Alexandre Augusto Pereira, Gorka Michal, Graf Alexander, Skirycz Aleksandra, Martinez-Seidel Federico, Zander Kerstin, Kopka Joachim, Beine-Golovchuk Olga
Max Planck Institute of Molecular Plant Physiology, 14476 Potsdam-Golm, Germany.
School of BioSciences, University of Melbourne, Melbourne, VIC 3010, Australia.
Plants (Basel). 2020 Jul 14;9(7):892. doi: 10.3390/plants9070892.
Conventional preparation methods of plant ribosomes fail to resolve non-translating chloroplast or cytoplasmic ribosome subunits from translating fractions. We established preparation of these ribosome complexes from leaf, root, and seed tissues by optimized sucrose density gradient centrifugation of protease protected plant extracts. The method co-purified non-translating 30S and 40S ribosome subunits separated non-translating 50S from 60S subunits, and resolved assembled monosomes from low oligomeric polysomes. Combining ribosome fractionation with microfluidic rRNA analysis and proteomics, we characterized the rRNA and ribosomal protein (RP) composition. The identity of cytoplasmic and chloroplast ribosome complexes and the presence of ribosome biogenesis factors in the 60S-80S sedimentation interval were verified. In vivo cross-linking of leaf tissue stabilized ribosome biogenesis complexes, but induced polysome run-off. Omitting cross-linking, the established paired fractionation and proteome analysis monitored relative abundances of plant chloroplast and cytoplasmic ribosome fractions and enabled analysis of RP composition and ribosome associated proteins including transiently associated biogenesis factors.
植物核糖体的传统制备方法无法从翻译组分中分离出非翻译的叶绿体或细胞质核糖体亚基。我们通过对蛋白酶保护的植物提取物进行优化的蔗糖密度梯度离心，从叶片、根和种子组织中建立了这些核糖体复合物的制备方法。该方法共纯化了非翻译的30S和40S核糖体亚基，将非翻译的50S与60S亚基分离，并从低聚多核糖体中分离出组装好的单核糖体。将核糖体分级分离与微流控rRNA分析和蛋白质组学相结合，我们对rRNA和核糖体蛋白（RP）组成进行了表征。验证了细胞质和叶绿体核糖体复合物的身份以及60S - 80S沉降区间中核糖体生物发生因子的存在。叶片组织的体内交联稳定了核糖体生物发生复合物，但诱导了多核糖体解离。省略交联步骤后，所建立的配对分级分离和蛋白质组分析监测了植物叶绿体和细胞质核糖体组分的相对丰度，并能够分析RP组成以及核糖体相关蛋白，包括瞬时相关的生物发生因子。
