Abstract
Identification of all the protein components of a plastid (chloroplast) ribosomal 30 S subunit has been achieved, using two-dimensional gel electropholesis, high performance liquid chromatography purification, N-terminal sequencing, polymerase chain reaction-based screening of cDNA library, nucleotide sequencing, and mass spectrometry (electrospray ionization, matrix-assisted laser desorption/ionization time-of-flight, and reversed-phase HPLC coupled with electrospray ionization mass spectrometry). 25 proteins were identified, of which 21 are orthologues of all Escherichia coli 30 S ribosomal proteins (S1-S21), and 4 are plastid-specific ribosomal proteins (PSRPs) that have no homologues in the mitochondrial, archaebacterial, or cytosolic ribosomal protein sequences in data bases. 12 of the 25 plastid 30 S ribosomal proteins (PRPs) are encoded in the plastid genome, whereas the remaining 13 are encoded by the nuclear genome. Post-translational transit peptide cleavage sites for the maturation of the 13 cytosolically synthesized PRPs, and post-translational N-terminal processing in the maturation of the 12 plastid synthesized PRPs are described. Post-translational modifications in several PRPs were observed: alpha-N-acetylation of S9, N-terminal processings leading to five mature forms of S6 and two mature forms of S10, C-terminal and/or internal modifications in S1, S14, S18, and S19, leading to two distinct forms differing in mass and/or charge (the corresponding modifications are not observed in E. coli). The four PSRPs in spinach plastid 30 S ribosomal subunit (PSRP-1, 26.8 kDa, pI 6.2; PSRP-2, 21.7 kDa, pI 5.0; PSRP-3, 13.8 kDa, pI 4.9; PSRP-4, 5.2 kDa, pI 11.8) comprise 16% (67.6 kDa) of the total protein mass of the 30 S subunit (429.3 kDa). PSRP-1 and PSRP-3 show sequence similarities with hypothetical photosynthetic bacterial proteins, indicating their possible origins in photosynthetic bacteria. We propose the hypothesis that PSRPs form a "plastid translational regulatory module" on the 30 S ribosomal subunit structure for the possible mediation of nuclear factors on plastid translation.
Highlights
Identification of all the protein components of a plastid ribosomal 30 S subunit has been achieved, using two-dimensional gel electropholesis, high performance liquid chromatography purification, N-terminal sequencing, polymerase chain reactionbased screening of cDNA library, nucleotide sequencing, and mass spectrometry. 25 proteins were identified, of which 21 are orthologues of all Escherichia coli 30 S ribosomal proteins (S1–S21), and 4 are plastid-specific ribosomal proteins (PSRPs) that have no homologues in the mitochondrial, archaebacterial, or cytosolic ribosomal protein sequences in data bases. 12 of the 25 plastid 30 S ribosomal proteins (PRPs) are encoded in the plastid genome, whereas the remaining 13 are encoded by the nuclear genome
Post-translational modifications in several plastid S ribosomal proteins (PRPs) were observed: ␣-N-acetylation of S9, N-terminal processings leading to five mature forms of S6 and two mature forms of S10, C-terminal and/or internal modifications in S1, S14, S18, and S19, leading to two distinct forms differing in mass and/or charge
Proteins of Chloroplast Ribosomal 30 S Subunit—To identify all the PRPs in pure chloroplast 30 S ribosomal subunit, spinach chloroplast 70 S ribosomes were first purified on a zonal gradient and run on a second dissociating zonal gradient to obtain 30 and 50 S subunits
Summary
IDENTIFICATION OF ALL THE PROTEINS IN THE 30 S SUBUNIT OF AN ORGANELLE RIBOSOME (CHLOROPLAST)*. 25 proteins were identified, of which 21 are orthologues of all Escherichia coli 30 S ribosomal proteins (S1–S21), and 4 are plastid-specific ribosomal proteins (PSRPs) that have no homologues in the mitochondrial, archaebacterial, or cytosolic ribosomal protein sequences in data bases. Two chloroplast-specific ribosomal proteins (PSRPs) in the 30 S ribosomal subunit with no obvious homology to any E. coli ribosomal proteins have been previously identified (14 –16). A summary of the results from this study are presented below, including identification of all the proteins in the plastid 30 S subunit, post-translational modifications, and identification of two additional novel PSRPs. A corresponding study of the plastid 50 S subunit is presented in the accompanying paper [54]
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