Dynamic protein composition of Arabidopsis thaliana cytosolic ribosomes in response to sucrose feeding as revealed by label free MS^E proteomics

Abstract

Cytosolic ribosomes are among the largest multisubunit cellular complexes. Arabidopsis thaliana ribosomes consist of 79 different ribosomal proteins (r-proteins) that each are encoded by two to six (paralogous) genes. It is unknown whether the paralogs are incorporated into the ribosome and whether the relative incorporation of r-protein paralogs varies in response to environmental cues. Immunopurified ribosomes were isolated from A. thaliana rosette leaves fed with sucrose. Trypsin digested samples were analyzed by qTOF-LC-MS using both MS(E) and classical MS/MS. Peptide features obtained by using these two methods were identified using MASCOT and Proteinlynx Global Server searching the theoretical sequences of A. thaliana proteins. The A. thaliana genome encodes 237 r-proteins and 69% of these were identified with proteotypic peptides for most of the identified proteins. These r-proteins were identified with average protein sequence coverage of 32% observed by MS(E) . Interestingly, the analysis shows that the abundance of r-protein paralogs in the ribosome changes in response to sucrose feeding. This is particularly evident for paralogous RPS3aA, RPS5A, RPL8B, and RACK1 proteins. These results show that protein synthesis in the A. thaliana cytosol involves a heterogeneous ribosomal population. The implications of these findings in the regulation of translation are discussed.

Mechanism of Cytoplasmic mRNA Translation

“…Ribosomes purified by differential centrifugation yielded 31-33 of the putative 40S RPs and 48 of the putative 60S RPs, respectively Giavalisco et al, 2005;Carroll et al, 2008;Piques et al, 2009;Turkina et al, 2011;Carroll, 2013). An analysis performed on ribosomes captured by immunopurification, thereby limiting contamination by organellar ribosomes and other co-sedimenting complexes, confirmed products from 204 of the estimated 232 functional RP genes of A. thaliana based on two or more prototypic peptides (Hummel et al, 2012). This corresponded to 64 of the 80 putative RP families and included RACK1.…”

“…The stereotype of a ribosome is that all are the same, but there is some evidence of specialized differences in cytosolic ribosomes of Arabidopsis and other plants that may contribute to the regulation of translation (Horiguchi et al, 2012;Hummel et al, 2012). Ribosome heterogeneity is predicted to be the con- RPSaB At3g04770 …”

“…a RP genes that have not been identified as pseudogenes (Barakat et al, 2001;Hummel et al, 2012; The Arabidopsis Information Resource, October 2014). Proteomic studies using 1D and 2D mass spectrometry have obtained evidence for the product of a least one paralog of each RP family in purified ribosomes, with the exception of the small (~3.5 kDa) and highly basic RPL41 (reviewed by Carroll, 2013).…”

Label-Free Protein Quantification for Plant Golgi Protein Localization and Abundance

“…Recently, Helm et al (2014) applied the LC-IMS-MS E with Top-3 quantification to quantify the Arabidopsis chloroplast stroma proteome, allowing quantitative modeling of chloroplast metabolism. Two other works used the LC-MS E method to assess the quantitative changes of cytosolic ribosomal proteins in response to Suc feeding and the extracellular proteome in response to salicylic acid (Cheng et al, 2009;Hummel et al, 2012).…”

RpL22e, but not RpL22e-like-PA, is SUMOylated and localizes to the nucleoplasm ofDrosophilameiotic spermatocytes

“…Many highly similar or identical Rp genes (demonstrated most notably in Saccharomyces cerevisiae and in Arabidopsis thaliana) appear to encode paralogues with functionally-distinct roles in several cellular, molecular or developmental pathways in response to different environmental cues. [1][2][3] These discoveries have stimulated an interest in determining if ribosome heterogeneity, in this case defined by Rp paralogue interchangeability, has an impact on translational regulation capacity (for a review, see ref. 4).…”