Preparation and Proteomic Analysis of Chloroplast Ribosomes [chapter]

Kenichi Yamaguchi
2011 Msphere  
Proteomics of chloroplast ribosomes in spinach and Chlamydomonas revealed unique protein composition and structures of plastid ribosomes. These studies have suggested the presence of some ribosomal proteins unique to plastid ribosomes which may be involved in plastid-unique translation regulation. Considering the strong background of genetic analysis and molecular biology in Arabidopsis, the in-depth proteomic characterization of Arabidopsis plastid ribosomes would facilitate further
more » ... further understanding of plastid translation in higher plants. Here, I describe simple and rapid methods for the preparation of plastid ribosomes from Chlamydomonas and Arabidopsis using sucrose gradients. I also describe purity criteria and methods for yield estimation of the purified plastid ribosomes and subunits, methods for the preparation of plastid ribosomal proteins, as well as the identification of some Arabidopsis plastid ribosomal proteins by matrix-assisted laser desorption/ionization mass spectrometry. Chlamydomonas (7, 8). Since two landmark discoveries published in 1962, of chloroplast ribosomes from spinach by Lyttleton (9) and of chloroplast DNA from Chlamydomonas by Ris and Plaut (10), the translational apparatus in chloroplasts has been mainly studied in spinach and Chlamydomonas; reviewed in (2, 11-13). Proteomic characterizations of plastid ribosomes from spinach (14-16) and Chlamydomonas (17, 18) have revealed that plastid ribosomes contain some plastid-specific ribosomal proteins (PSRPs) in addition to bacterial orthologs. These proteomic studies also revealed differences in protein composition and the primary structure of each ribosomal protein between higher plants and green algae. Recent cryo-electron microscopy of plastid ribosomes from spinach (19) and Chlamydomonas (20) has visualized the 3D-localization of PSRPs and plastid-specific domains in the ribosomes, suggesting their involvement in translation regulation. Although functional analyses of some PSRPs (PSRP-1 in spinach and PSRP-7 in Chlamydomonas) have been reported (21, 22), the physiological roles of another five PSRPs (PSRP-2 to PSRP-6) remain unclear. In addition, post-translational modifications, which may also affect translational activity of plastid ribosomes, remain to be elucidated. Although advanced proteomic analyses of Arabidopsis cytoplasmic 80S ribosomes have been performed (23-25), proteomic characterization of Arabidopsis plastid ribosomes
doi:10.1007/978-1-61779-237-3_13 pmid:21863447 fatcat:im6xpuikpfbkvadcz7d3qxawy4