Comprehensive Analysis of Phosphorylated Proteins of Escherichia coli Ribosomes

Abstract

Phosphorylation of bacterial ribosomal proteins has been known for decades; however, there is still very limited information available on specific locations of the phosphorylation sites in ribosomal proteins and the role they might play in protein synthesis. In this study, we have mapped the specific phosphorylation sites in twenty-four E. coli ribosomal proteins by tandem mass spectrometry. Specific detection of phosphorylation was achieved by either phosphorylation specific visualization techniques, ProQ staining and antibodies for phospho-Ser, Thr, and Tyr, or by mass spectrometry equipped with a capability to detect addition and the loss of the phosphate moiety. Enrichment by immobilized metal affinity and/or strong cation exchange chromatography was used to improve the success of detection of the low abundance phosphopeptides. We found the small subunit (30S) proteins S3, S4, S5, S7, S11, S12, S13, S18, and S21 and the large subunit (50S) proteins L1, L2, L3, L5, L6, L7/L12, L13, L14, L16, L18, L19, L21, L22, L28, L31 to be phosphorylated at one or more residues. Potential roles for each specific site in ribosome function were deduced through careful evaluation of the given site of the phosphorylation in 3D-crystal structure models of ribosomes and the previous mutational studies of E. coli ribosomal proteins.

Identification and characterization of CHCHD1, AURKAIP1, and CRIF1 as new members of the mammalian mitochondrial ribosome

“…The pieces were processed by performing in-gel tryptic digestion using methods previously established in our laboratory (Miller et al, 2008, 2009; Soung et al, 2009). Tryptic digests were analyzed by capillary liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry (capLC-MS/MS).…”

Bacterial tyrosine kinases: evolution, biological function and structural insights

“…Upon examination of BYmediated protein phosphorylation sites, no clear-cut recognition motifs emerged in the immediate surroundings of the phosphorylated tyrosines. Moreover, if one considers the number of tyrosine-phosphorylation sites detected in recent phosphoproteomic studies (table 1), the conservation of phosphotyrosine sites is nearly non-existent [84,[91][92][93][94][95][96][97][98][99]. The existence of a specific phosphorylation motif is therefore unlikely in proteins phosphorylated by BY-kinases, and it seems likely that they recognize structural motifs distant from the phosphorylated tyrosine.…”

The Role of Bacterial Protein Tyrosine Phosphatases in the Regulation of the Biosynthesis of Secreted Polysaccharides

“…Whereas a number have been identified and discussed above, it is likely that there are a large number of additional targets. In recent times, phosphoproteomic studies carried out across a range of bacterial species have led to the discovery of a range of proteins phosphorylated on serine, threonine, and tyrosine (56,59,94,101). Whereas those phosphorylated on tyrosine constituted the smallest group, this is due, in part, to the fact that the methods used in these studies favor identification of more heavily phosphorylated amino acids, with tyrosine normally phosphorylated at low percentages.…”

“…Indeed, increased identification of PY has already been evident in investigations of E. coli. Whereas a phosphoproteomic analysis of the whole cell led to the identification of only seven tyrosines phosphorylated (55), a more defined study of only ribosomal-binding proteins found that 11 of these proteins were tyrosine phosphorylated (94). Furthermore, a recent study utilizing methods to solely investigate the tyrosine phosphoproteome of E. coli found a total of 342 tyrosine phosphorylated proteins (38).…”