Isolation of Cytosolic Ribosomes Associated with Plant Mitochondria and Chloroplasts

Excluding the few dozen proteins encoded by the chloroplast and mitochondrial genomes, the majority of plant cell proteins are synthesized by cytosolic ribosomes. Most of these nuclear-encoded proteins are then targeted to specific cell compartments thanks to localization signals present in their amino acid sequence. These signals can be specific amino acid sequences known as transit peptides, or post-translational modifications, ability to interact with specific proteins or other more complex regulatory processes. Furthermore, in eukaryotic cells, protein synthesis can be regulated so that certain proteins are synthesized close to their destination site, thus enabling local protein synthesis in specific compartments of the cell. Previous studies have revealed that such locally translating cytosolic ribosomes are present in the vicinity of mitochondria and emerging views suggest that localized translation near chloroplasts could also occur. However, in higher plants, very little information is available on molecular mechanisms controlling these processes and there is a need to characterize cytosolic ribosomes associated with organelles membranes. To this goal, this protocol describes the purification of higher plant chloroplast and mitochondria and the organelle-associated cytosolic ribosomes.