Chloroplast biogenesis involves spatial coordination of nuclear and organellar gene expression in Chlamydomonas.

The localization of translation can direct the polypeptide product to the proper intracellular compartment. Our results reveal translation by cytosolic ribosomes on a domain of the chloroplast envelope in the unicellular green alga Chlamydomonas (Chlamydomonas reinhardtii). We show that this envelope domain of isolated chloroplasts retains translationally active ribosomes and mRNAs encoding chloroplast proteins. This domain is aligned with localized translation by chloroplast ribosomes in the translation zone, a chloroplast compartment where photosystem subunits encoded by the plastid genome are synthesized and assembled. Roles of localized translation in directing newly synthesized subunits of photosynthesis complexes to discrete regions within the chloroplast for their assembly are suggested by differences in localization on the chloroplast of mRNAs encoding either subunit of the light-harvesting complex II or the small subunit of Rubisco. Transcription of the chloroplast genome is spatially coordinated with translation, as revealed by our demonstration of a subpopulation of transcriptionally active chloroplast nucleoids at the translation zone. We propose that the expression of chloroplast proteins by the nuclear-cytosolic and organellar genetic systems is organized in spatially aligned subcompartments of the cytoplasm and chloroplast to facilitate the biogenesis of the photosynthetic complexes.

An RNA granule for translation quality control in Saccharomyces cerevisiae.

Cytoplasmic RNA granules compartmentalize phases of the translation cycle in eukaryotes. We previously reported the localization of oxidized RNA to cytoplasmic foci called oxidized RNA bodies (ORBs) in human cells. We show here that ORBs are RNA granules in Saccharomyces cerevisiae. Several lines of evidence support a role for ORBs in the compartmentalization of no-go decay and ribosome quality control, the translation quality control pathways that recognize and clear aberrant mRNAs, including those with oxidized bases. Translation is required by these pathways and ORBs. Translation quality control factors localize to ORBs. A substrate of translation quality control, a stalled mRNA-ribosome-nascent-chain complex, localizes to ORBs. Translation quality control mutants have altered ORB numbers, sizes or both. In addition, we identify 68 ORB proteins by immunofluorescence staining directed by proteomics, which further support their role in translation quality control and reveal candidate new factors for these pathways.

Pyrenoid functions revealed by proteomics in Chlamydomonas reinhardtii.

Organelles are intracellular compartments which are themselves compartmentalized. Biogenic and metabolic processes are localized to specialized domains or microcompartments to enhance their efficiency and suppress deleterious side reactions. An example of intra-organellar compartmentalization is the pyrenoid in the chloroplasts of algae and hornworts. This microcompartment enhances the photosynthetic CO2-fixing activity of the Calvin-Benson cycle enzyme Rubisco, suppresses an energetically wasteful oxygenase activity of Rubisco, and mitigates limiting CO2 availability in aquatic environments. Hence, the pyrenoid is functionally analogous to the carboxysomes in cyanobacteria. However, a comprehensive analysis of pyrenoid functions based on its protein composition is lacking. Here we report a proteomic characterization of the pyrenoid in the green alga Chlamydomonas reinhardtii. Pyrenoid-enriched fractions were analyzed by quantitative mass spectrometry. Contaminant proteins were identified by parallel analyses of pyrenoid-deficient mutants. This pyrenoid proteome contains 190 proteins, many of which function in processes that are known or proposed to occur in pyrenoids: e.g. the carbon concentrating mechanism, starch metabolism or RNA metabolism and translation. Using radioisotope pulse labeling experiments, we show that pyrenoid-associated ribosomes could be engaged in the localized synthesis of the large subunit of Rubisco. New pyrenoid functions are supported by proteins in tetrapyrrole and chlorophyll synthesis, carotenoid metabolism or amino acid metabolism. Hence, our results support the long-standing hypothesis that the pyrenoid is a hub for metabolism. The 81 proteins of unknown function reveal candidates for new participants in these processes. Our results provide biochemical evidence of pyrenoid functions and a resource for future research on pyrenoids and their use to enhance agricultural plant productivity. Data are available via ProteomeXchange with identifier PXD004509.

Localized control of oxidized RNA.

The oxidation of biological molecules by reactive oxygen species (ROS) can render them inactive or toxic. This includes the oxidation of RNA, which appears to underlie the detrimental effects of oxidative stress, aging and certain neurodegenerative diseases. Here, we investigate the management of oxidized RNA in the chloroplast of the green alga Chlamydomonas reinhardtii. Our immunofluorescence microscopy results reveal that oxidized RNA (with 8-hydroxyguanine) is localized in the pyrenoid, a chloroplast microcompartment where CO2 is assimilated by the Calvin cycle enzyme Rubisco. Results of genetic analyses support a requirement for the Rubisco large subunit (RBCL), but not Rubisco, in the management of oxidized RNA. An RBCL pool that can carry out such a 'moonlighting' function is revealed by results of biochemical fractionation experiments. We also show that human (HeLa) cells localize oxidized RNA to cytoplasmic foci that are distinct from stress granules, processing bodies and mitochondria. Our results suggest that the compartmentalization of oxidized RNA management is a general phenomenon and therefore has some fundamental significance.