Chloroplast protein targeting involves localized translation in Chlamydomonas

The compartmentalization of eukaryotic cells requires that newly synthesized proteins be targeted to the compartments in which they function. In chloroplasts, a few thousand proteins function in photosynthesis, expression of the chloroplast genome, and other processes. Most chloroplast proteins are synthesized in the cytoplasm, imported, and then targeted to a specific chloroplast compartment. The remainder are encoded by the chloroplast genome, synthesized within the organelle, and targeted by mechanisms that are only beginning to be elucidated. We used fluorescence confocal microscopy to explore the targeting mechanisms used by several chloroplast proteins in the green alga Chlamydomonas. These include the small subunit of ribulose bisphosphate carboxylase (rubisco) and the light-harvesting complex II (LHCII) subunits, which are imported from the cytoplasm, and 2 proteins synthesized in the chloroplast: the D1 subunit of photosystem II and the rubisco large subunit. We determined whether the targeting of each protein involves localized translation of the mRNA that encodes it. When this was the case, we explored whether the targeting sequence was in the nascent polypeptide or in the mRNA, based on whether the localization was translation-dependent or -independent, respectively. The results reveal 2 novel examples of targeting by localized translation, in LHCII subunit import and the targeting of the rubisco large subunit to the pyrenoid. They also demonstrate examples of each of the three known mechanisms - posttranslational, cotranslational (signal recognition particle-mediated), and mRNA-based - in the targeting of specific chloroplast proteins. Our findings can help guide the exploration of these pathways at the biochemical level.