de Jong Mark, van Breukelen Bas, Wittink Floyd R, Menke Frank L H, Weisbeek Peter J, Van den Ackerveken Guido
Molecular Genetics Group, Department of Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Plant J. 2006 May;46(4):708-21. doi: 10.1111/j.1365-313X.2006.02724.x.
Membrane-associated, integral membrane and secreted proteins are of key importance in many cellular processes. For most of the 28,952 predicted proteins in Arabidopsis, the actual subcellular localization has not been demonstrated experimentally. So far, their potential membrane-association has been deduced from algorithms that predict transmembrane domains and signal peptides. However, the comprehensiveness and accuracy of these algorithms is still limited. The majority of membrane-associated and secreted proteins is synthesized on membrane-bound polysomes. Therefore, the isolation and characterization of mRNA associated with membrane-bound polysomes offers an experimental tool for the genome-wide identification of these proteins. Here we describe an efficient method to isolate mRNA from membrane-bound polysomes and report on the validation of the method to enrich for transcripts encoding membrane-associated and secreted proteins. The sensitivity and reproducibility of the isolation method was investigated by DNA microarray analysis. Pearson correlations between transcript levels obtained from three replicate isolations showed that the method is highly reproducible. A significant enrichment for mRNAs encoding proteins containing predicted transmembrane domains and signal peptides was observed in the membrane-bound polysomal fraction. In this fraction, 301 transcripts were classified by gene ontologies as 'cellular component unknown', and potentially encode previously unrecognized secreted or membrane-associated proteins.
膜相关蛋白、整合膜蛋白和分泌蛋白在许多细胞过程中至关重要。对于拟南芥中预测的28952种蛋白质中的大多数，其实际亚细胞定位尚未通过实验证明。到目前为止，它们潜在的膜关联性是通过预测跨膜结构域和信号肽的算法推断出来的。然而，这些算法的全面性和准确性仍然有限。大多数膜相关蛋白和分泌蛋白是在膜结合多核糖体上合成的。因此，分离和鉴定与膜结合多核糖体相关的mRNA为全基因组鉴定这些蛋白质提供了一种实验工具。在这里，我们描述了一种从膜结合多核糖体中分离mRNA的有效方法，并报告了该方法用于富集编码膜相关蛋白和分泌蛋白的转录本的验证情况。通过DNA微阵列分析研究了分离方法的灵敏度和可重复性。三次重复分离获得的转录本水平之间的皮尔逊相关性表明该方法具有高度可重复性。在膜结合多核糖体部分观察到编码含有预测跨膜结构域和信号肽的蛋白质的mRNA有显著富集。在这个部分，301个转录本通过基因本体分类为“细胞成分未知”，并可能编码以前未识别的分泌蛋白或膜相关蛋白。
