CHLOROPLAST RIBOSOME ASSOCIATED Supports Translation under Stress and Interacts with the Ribosomal 30S Subunit

Abstract

Chloroplast ribosomes, which originated from cyanobacteria, comprise a large subunit (50S) and a small subunit (30S) containing ribosomal RNAs (rRNAs) and various ribosomal proteins. Genes for many chloroplast ribosomal proteins, as well as proteins with auxiliary roles in ribosome biogenesis or functioning, reside in the nucleus. Here, we identified Arabidopsis () CHLOROPLAST RIBOSOME ASSOCIATED (CRASS), a member of the latter class of proteins, based on the tight coexpression of its mRNA with transcripts for nucleus-encoded chloroplast ribosomal proteins. CRASS was acquired during the evolution of embryophytes and is localized to the chloroplast stroma. Loss of CRASS results in minor defects in development, photosynthetic efficiency, and chloroplast translation activity under controlled growth conditions, but these phenotypes are greatly exacerbated under stress conditions induced by the translational inhibitors lincomycin and chloramphenicol or by cold treatment. The CRASS protein comigrates with chloroplast ribosomal particles and coimmunoprecipitates with the 16S rRNA and several chloroplast ribosomal proteins, particularly the plastid ribosomal proteins of the 30S subunit (PRPS1 and PRPS5). The association of CRASS with PRPS1 and PRPS5 is independent of rRNA and is not detectable in yeast two-hybrid experiments, implying that either CRASS interacts indirectly with PRPS1 and PRPS5 via another component of the small ribosomal subunit or that it recognizes structural features of the multiprotein/rRNA particle. CRASS plays a role in the biogenesis and/or stability of the chloroplast ribosome that becomes critical under certain stressful conditions when ribosomal activity is compromised.

The Arabidopsis Protein CGL20 Is Required for Plastid 50S Ribosome Biogenesis

“…5B). Pronounced effects at low temperature have also been observed in several other Arabidopsis mutants that are impaired in chloroplast protein synthesis (Sugimoto et al, 2004;Paieri et al, 2018;Pulido et al, 2018) as well as in bacterial strains with defects in ribosome biogenesis (Davis and Williamson, 2017). Thus, alternative LSU assembly pathways might exist that bypass AtCGL20 function.…”

“…), and subjected to autoradiography. Abundant signals were identified as RbcL, CP43/CP47, and D1/D2 according to Rü hle et al 2014and Pulido et al (2018). C, RNA gel-blot hybridization of fractions obtained following Suc density-gradient centrifugation under polysome-preserving conditions.…”

“…Some of these have been shown to mediate similar functions in chloroplasts (for review, see Bohne, 2014;Liu et al, 2015;Jeon et al, 2017;Janowski et al, 2018). Despite the dual genetic origin of plastid RPs, which necessitates spatiotemporal orchestration of RP assembly, and significant structural differences with respect to bacterial ribosomes, few chloroplast-specific ribosome biogenesis factors have been identified to date (Bohne, 2014;Wang et al, 2016;Meurer et al, 2017;Paieri et al, 2018;Pulido et al, 2018).…”

A pan-plant protein complex map reveals deep conservation and novel assemblies

“…We found several instances where a top-scoring interaction to an uncharacterized protein could be con irmed in the literature, serving as additional positive controls. For example, the most con ident interactor with the recently characterized Arabidopsis protein AT5G14910 is the chloroplast ribosome protein RPS1; the interaction was independently con irmed in a recent study (Pulido et al, 2018) . We also observed instances of interactions between proteins catalyzing consecutive enzymatic reactions, such as a novel interaction between OXP1 and GEP, enzymes catalyzing the last two steps of glutathione degradation.…”

Co-expressed subunits of dual genetic origin define a conserved supercomplex mediating essential protein import into chloroplasts

“…Indeed, chloroplast tic214 and orf2971 are co-expressed with nuclear TIC20 during the Chlamydomonas diurnal cycle, suggesting their common regulation. Thus, in addition to the photosynthetic complexes and the chloroplast ribosome(86)(87)(88), the chloroplast translocon emerges as yet another example of coordinated gene expression between the chloroplast and nuclear compartment.In Arabidopsis, TIC and TOC are linked together through Tic236, an integral inner-membrane protein that directly binds to Toc75 via its C-terminal domain protruding into the intermembrane space (89). Although BLAST analyses identify an algal orthologue of Tic236 (Cre05.g243150), we did not detect this protein in our experiments or co-expression studies.…”