The Plastid Ribosomal Proteins

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 reactionbased screening of cDNA library, nucleotide sequencing, and mass spectrometry (electrospray ionization, matrixassisted 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: ␣-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.

Proteomics Uncovers Proteins Interacting Electrostatically with Thioredoxin in Chloroplasts

“…In the Calvin cycle, the rubisco large subunit assumes a quaternary structure with the small subunit in yielding a functional enzyme, and phosphoglycerate kinase forms a complex with glyceraldehyde 3-phosphate dehydrogenase -an association that likely results in substrate channeling (Wang et al 1996). Similarly, the 24 and 41 kDa RNA binding proteins as well as ribosomal proteins S1, S5, L4 and L21 are known to form complexes with other thioredoxin target proteins -namely, the 28 kDa ribonucleoprotein and ribosomal protein S30, respectively (Hayes et al 1996;Yamaguchi et al 2000). Finally, the chloroplast ATP synthase alpha subunit may co-elute with the enzyme's gamma subunit, an established thioredoxin target.…”

Cryo-EM study of the spinach chloroplast ribosome reveals the structural and functional roles of plastid-specific ribosomal proteins

“…In addition, the chloro-ribosome possesses six nonorthologous proteins, which are termed ''plastid-specific ribosomal proteins'' (PSRPs): four of these proteins (PSRP1-4) are associated with the 30S subunit, and two (PSRP5 and PSRP6) are associated with the 50S subunit. The larger size of the PRPs as well as the presence of six PSRPs leads to a significantly altered protein/RNA mass ratio of 2/3 in the case of chlororibosomes, as compared with 1/3 for E. coli (5,6). The higher protein content of chloro-ribosomes, and especially the presence of PSRPs, has prompted the suggestion that chloroplasts have evolved specific proteins that play unique functional roles during chloroplast translation, such as the light-dependent stimulation of protein synthesis (5-7).…”

“…The ribosomal RNAs (rRNAs) are similar in length and exhibit relatively few differences [see supporting information (SI) Figs. [6][7][8]. The 16S rRNAs of the Escherichia coli 30S subunit has 1,542 nt, whereas the spinach chloro-30S subunit is slightly smaller, containing 1,491 nt.…”

“…Proteomic studies reveal that the chloro-ribosomes possess in total 58 ribosomal proteins, 25 and 33 proteins in the 30S and 50S subunits, respectively (5,6), and have orthologs to all 52 E. coli ribosomal proteins, with the exception of L25 and L30. Plastid ribosomal proteins (PRPs) generally are larger than their E. coli counterparts, containing extensions predominantly at the N and C termini.…”

The Plastid Ribosomal Proteins

“…Post-translational modifications are found in many E. coli RPs, although a modification in L16 (Arg 81 ) remains yet to be characterized (see "Results" for plastid L16). We have recently identified all the RPs in spinach plastid 30 S ribosomal subunit, including all its PSRPs and many post-translational modifications (83). The number of RPs in plastid 50 S subunits has so far only been estimated (ϳ35-39; reviewed in Ref.…”