Characterization of chloroplast protein import in red alga, Cyanidioschyzon merolae

概要

Chloroplasts are green-colored organelles with complex structures, found only in plants and algae. Energy is essential for growth and reproduction in both animals and plants. While animals consume food to produce energy, plants and algae use chloroplasts to produce the energy they need directly from the sun. This may be a good justification for the study of chloroplasts. The chloroplasts in photosynthetic eukaryotes originated from the cyanobacterium-like endosymbiont and still retain their own genomes. Only about 100 proteins are encoded by the chloroplast genomes, whereas more than 2000 different kinds of chloroplast proteins are encoded by the nuclear genomes, synthesized in the cytosol, and then transported into the chloroplasts. TOC and TIC, translocons at the outer and inner envelope membranes of chloroplasts, respectively, play an essential role in protein transport. Our team previously identified a novel TIC complex consisting of Tic214(Ycf1)/Tic100/Tic56/Tic20 from Arabidopsis thaliana as a main TIC complex and also recently identified a completely novel Ycf2/FtsHi ATP-driven motor complex which associates with the TIC complex. The TIC and the associated motor complex are widely distributed among green lineages including plants and green alga. Red algae diverged quite early from the green lineages during the evolution of photosynthetic eukaryotes. The chloroplast protein transport system of red algae has not yet been studied. Moreover, in the genome of red algae, we could not find orthologs for some of well-known TOC/TIC components except for some central ones. Thus, red alga might retain an ancestral type of chloroplast protein transport system. I aimed to identify and characterize this system in Cyanidioschyzon merolae 10D, a model organism of red algae.

In this study, I firstly attempted to establish the in vitro chloroplast protein import system using intact chloroplasts isolated from the red algal cells with purified model pre-proteins, which consist of a chloroplast-targeted preprotein of red algal origin and some tags for detection and purification. Although intact chloroplasts were properly isolated, proteins were not imported into chloroplasts. Therefore, we adopted an in vivo approach to characterize chloroplast protein import in this red alga. To this end, a C. merolae transformant inducibly expressing chloroplast-targeted GFP-fusion protein (Tp-GFP-3xFLAG) was used to purify translocation intermediates. Immunoprecipitation of induced cell lysates using anti-3xFLAG antibody identified Tic20 and FtsH as preprotein-interacting proteins during import. Besides those, a class of GTP-binding proteins, CMI311C and CMQ054C proteins (provisional names), distinct from well-known Toc34/Toc159 preprotein receptor GTPase in green lineages, were found associated with transporting preproteins. Additional experiments suggested that 311C and 054C proteins directly recognize the transit peptides of preproteins. While, in the C. merolae genomic database, CMP284C and CMQ137C were tentatively annotated as Toc34/To159 homologs, the above-mentioned in vivo analysis of preprotein-associated proteins failed to detect them. These data suggest that the Tic20-centered TIC complex and the FtsH import motor likely participate in chloroplast protein import in red algae as in the green lineage. However, TOC complex and cytosolic events related to chloroplast protein import in red algae may be quite different from those of the green lineages, which involve the distinct class of GTP binding proteins. Altogether, the relevance of this study is underscored by the identification of the early chloroplast protein import pathway, it is the first to identify the chloroplast protein import pathway using red algae. Further studies will be needed to elucidate more detailed mechanisms of chloroplast protein import in red alga.