title: Proteomic LC-MS analysis of Arabidopsis cytosolic ribosomes: Identification of ribosomal protein paralogs and re-annotation of the ribosomal protein genes.
abstract: 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 classic sucrose cushion centrifugation-based protocol from plants of different developmental stages. Both 1D and 2D LC-MS(E) 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-protein 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.
  
  
  CONCLUSIONS
  Translation is one of the most energy demanding processes in a living cell and is therefore carefully regulated. Translational activity is tightly linked to growth control and growth regulating mechanism. Recently established translational profiling technologies, including the profiling of mRNAs associated with polysomes and the mapping of ribosome footprints on mRNAs, have revealed that the expression of gene expression is often fine-tuned by differential translation of gene transcripts. The eukaryotic ribosome, the hub of these important processes, consists of close to eighty different proteins (depending on species) and four large RNAs assembled into two highly conserved subunits. In plants and to lesser extent in yeast, the r-proteins are encoded by more than one actively transcribed gene. As r-protein gene paralogs frequently do not encode identical proteins and are regulated by growth conditions and development, in vivo ribosomes are heterogeneous in their protein content. The regulatory and physiological importance of this heterogeneity is unknown. Here, an improved annotation of the more than two hundred r-protein genes of Arabidopsis is presented that combines proteomic and advanced mRNA expression data. This proteomic investigation and re-annotation of Arabidopsis ribosomes establish a base for future investigations of translational control in plants.
authors.affiliation: Molecular Plant Physiology, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands; Center for Plant Cell Biology, Department of Botany and Plant Sciences, University of California, Riverside, CA 92521-0124, USA.
authors.forename: Maureen
authors.initials: M
authors.lastname: Hummel
authors.affiliation: Umeå Plant Science Center, Department of Plant Physiology, Umeå University, 90187, Umeå, Sweden; Institut Jean-Pierre Bourgin, UMR 1318 INRA AgroParisTech, Saclay Plant Sciences, F-78026 Versailles, France.
authors.forename: Thomas
authors.initials: T
authors.lastname: Dobrenel
authors.affiliation: BU Bioscience, Plant Research International, P.O. Box 619, 6700 AP Wageningen, The Netherlands.
authors.forename: Jan J H G
authors.initials: JJ
authors.lastname: Cordewener
authors.affiliation: Plateforme PAPPSO, UMR de Génétique Végétale, Ferme du Moulon, Gif sur Yvette, France.
authors.forename: Marlène
authors.initials: M
authors.lastname: Davanture
authors.affiliation: Institut Jean-Pierre Bourgin, UMR 1318 INRA AgroParisTech, Saclay Plant Sciences, F-78026 Versailles, France.
authors.forename: Christian
authors.initials: C
authors.lastname: Meyer
authors.affiliation: Molecular Plant Physiology, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands; Centre for BioSystems Genomics, P.O. Box 98, 6700 AB Wageningen, The Netherlands.
authors.forename: Sjef J C M
authors.initials: SJ
authors.lastname: Smeekens
authors.affiliation: Center for Plant Cell Biology, Department of Botany and Plant Sciences, University of California, Riverside, CA 92521-0124, USA.
authors.forename: Julia
authors.initials: J
authors.lastname: Bailey-Serres
authors.affiliation: Centre for BioSystems Genomics, P.O. Box 98, 6700 AB Wageningen, The Netherlands; Netherlands Proteomics Centre, P.O. Box 80082, 3508 TB Utrecht, The Netherlands.
authors.forename: Twan A H P
authors.initials: TA
authors.lastname: America
authors.affiliation: Molecular Plant Physiology, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands; Umeå Plant Science Center, Department of Plant Physiology, Umeå University, 90187, Umeå, Sweden. Electronic address: johannes.hanson@umu.se.
authors.forename: Johannes
authors.initials: J
authors.lastname: Hanson
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D020543:Proteome / Q000737:chemistry / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D020543:Proteome; D012269:Ribosomal Proteins
keywords: A. thaliana; DIA; Data-independent acquisition; LC–MS; Paralogs; Ribosomal protein; Ribosomes