Proteomic Characterization of the Chlamydomonas reinhardtii Chloroplast Ribosome

Abstract

We have conducted a proteomic analysis of the 70 S ribosome from the Chlamydomonas reinhardtii chloroplast. Twenty-seven orthologs of Escherichia coli large subunit proteins were identified in the 50 S subunit, as well as an ortholog of the spinach plastid-specific ribosomal protein-6. Several of the large subunit proteins of C. reinhardtii have short extension or insertion sequences, but overall the large subunit proteins are very similar to those of spinach chloroplast and E. coli. Two proteins of 38 and 41 kDa, designated RAP38 and RAP41, were identified from the 70 S ribosome that were not found in either of the ribosomal subunits. Phylogenetic analysis identified RAP38 and RAP41 as paralogs of spinach CSP41, a chloroplast RNA-binding protein with endoribonuclease activity. Overall, the chloroplast ribosome of C. reinhardtii is similar to those of spinach chloroplast and E. coli, but the C. reinhardtii ribosome has proteins associated with the 70 S complex that are related to non-ribosomal proteins in other species. In addition, the 30 S subunit contains unusually large orthologs of E. coli S2, S3, and S5 and a novel S1-type protein (Yamaguchi, K. et al., (2002) Plant Cell 14, 2957-2974). These additional proteins and domains likely confer functions used to regulate chloroplast translation in C. reinhardtii.In the chloroplast, where proteins of the photosynthetic apparatus and the carbon-fixing enzymes are synthesized, gene expression is primarily regulated during translation (1). Chloroplast translation has been thought to be similar to translation in bacterial systems, mainly because of similarities in ribosomal RNA and the sensitivity of chloroplast ribosomes to bacterial antibiotics. These similarities support the endosymbiotic theory that chloroplasts originated from a photosynthetic prokaryote, cyanobacteria (2, 3). It is now recognized that chloroplast gene expression and chloroplast translation are unique and quite different from bacterial systems (1, 4 -7). The chloroplast ribosome contains plastid-specific ribosomal proteins (PSRPs) 1 in addition to bacterial orthologs (5-7). It has been proposed that PSRPs may take part in the unique light-dependent aspects of chloroplast translation (8). Bacterial gene expression is strongly influenced by the rate of transcription, and translation and transcription are often closely coupled. In the chloroplast, transcription is often globally regulated, and mRNA accumulation can be unrelated to the rate of translation of a protein (reviewed in Refs. 1 and 9 -13). Translation of many chloroplast mRNAs is activated in response to light illumination, with little change in the corresponding mRNA levels (14 -19). A majority of the work on chloroplast translation has been carried out in the unicellular green alga, Chlamydomonas reinhardtii (reviewed in Refs. 13, 20, and 21), because it is amenable to both genetic and biochemical analysis (22,23). Identification of all of the proteins required for chloroplast translation in C. reinhardtii would facilitate our understand...

Analysis of the Phosphoproteome of Chlamydomonas reinhardtii Provides New Insights into Various Cellular Pathways

“…We also performed a comparative proteome analysis with already known subproteomes from the chloroplast 70S ribosome (33,34), thioredoxin interaction partners (18), the flagella (24), and the centriole (13). In this way, we were able to identify several proteins of these subproteomes that are subject to phosphorylation (Table 3).…”

Functional Differentiation of Bundle Sheath and Mesophyll Maize Chloroplasts Determined by Comparative Proteomics

“…In addition, searching the MS data against the maize genomic sequences (in AZM) identified a protein encoded by CF646019 (and AZM4_135414) similar to Arabidopsis CSP41, which was characterized as an mRNA binding protein in spinach chloroplasts with endoribonuclease activity (Yang et al, 1996) and is a homolog of C. reinhardtii RAP41 ribosome-associated protein (Yamaguchi et al, 2003). The expression of CSP41 (CF646019, AZM4_135414) shows a small preference for the BS chloroplasts (BS:M ratio ¼ 1.8-fold).…”

Plastoglobule Proteome

“…In Chlamydomonas, RAP38 copurifies with 70S chloroplast ribosomes (Yamaguchi et al, 2003). These proteins contain epimerase/dehydrase domains but their function is not clear.…”