Proteomic LC-MS analysis of Arabidopsis cytosolic ribosomes: Identification of ribosomal protein paralogs and re-annotation of the ribosomal protein genes
Hummel, M. ; Dobrenel, T. ; Cordewener, J.H.G. ; Davanture, M. ; Meyer, C. ; Smeekens, J.C.M. ; Bailey-Serres, J. ; America, A.H.P. ; Hanson, J. - \ 2015
Journal of Proteomics 128 (2015). - ISSN 1874-3919 - p. 436 - 449.
Arabidopsis thaliana cytosolic ribosomes are large complexes containing eighty-one distinct ribosomal proteins (r-proteins), four ribosomal RNAs (rRNA) and a plethora of associated (non-ribosomal) proteins. In plants, r-proteins of cytosolic ribosomes are each encoded by two to seven different expressed and similar genes, forming an r-protein family. Distinctions in the r-protein coding sequences of gene family members are a source of variation between ribosomes. We performed proteomic investigation of actively translating cytosolic ribosomes purified using both immunopurification and a classical sucrose cushion centrifugation-based protocol from plants of different developmental stages. Both 1D and 2D LC-MSE with data-independent acquisition as well as conventional data-dependent MS/MS procedures were applied. This approach provided detailed identification of 165 r-protein paralogs with high coverage based on proteotypic peptides. The detected r-proteins were the products of the majority (68%) of the 242 cytosolic r-proteins genes encoded by the genome. A total of 70 distinct r-proteins were identified. Based on these results and information from DNA microarray and ribosome footprint profiling studies a re-annotation of Arabidopsis r-proteins and genes is proposed. This compendium of the cytosolic r-protein proteome will serve as a template for future investigations on the dynamic structure and function of plant ribosomes.