Comprehensive analysis of phosphorylated proteins of Escherichia coli ribosomes.
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 24 Escherichia coli ribosomal proteins by tandem mass spectrometry. 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 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, and 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 phosphorylation sites in 3D-crystal structure models of ribosomes and the previous mutational studies of E. coli ribosomal proteins.
Soung
George Y
GY
Department of Biochemistry & Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Miller
Jennifer L
JL
Koc
Hasan
H
Koc
Emine C
EC
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000378:metabolism*; D015151:Immunoblotting / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010710:Phosphates / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D008670:Metals; D010710:Phosphates; D010748:Phosphopeptides