The two-membrane envelope is a defining feature of chloroplasts. Chloroplasts evolved from a Gram-negative cyanobacterial endosymbiont. During evolution, genes of the endosymbiont have been transferred to the host nuclear genome. Most chloroplast proteins are synthesized in the cytosol as higher-molecular-mass preproteins with an N-terminal transit peptide. Preproteins are transported into chloroplasts by the TOC and TIC (translocons at the outer- and inner-envelope membranes of chloroplasts, respectively) machineries1,2, but how TOC and TIC are assembled together is unknown. Here we report the identification of the TIC component TIC236; TIC236 is an integral inner-membrane protein that projects a 230-kDa domain into the intermembrane space, which binds directly to the outer-membrane channel TOC75. The knockout mutation of TIC236 is embryonically lethal. In TIC236-knockdown mutants, a smaller amount of the inner-membrane channel TIC20 was associated with TOC75; the amount of TOC–TIC supercomplexes was also reduced. This resulted in a reduced import rate into the stroma, though outer-membrane protein insertion was unaffected. The size and the essential nature of TIC236 indicate that—unlike in mitochondria, in which the outer- and inner-membrane translocons exist as separate complexes and a supercomplex is only transiently assembled during preprotein translocation3,4—a long and stable protein bridge in the intermembrane space is required for protein translocation into chloroplasts. Furthermore, TIC236 and TOC75 are homologues of bacterial inner-membrane TamB5 and outer-membrane BamA, respectively. Our evolutionary analyses show that, similar to TOC75, TIC236 is preserved only in plants and has co-evolved with TOC75 throughout the plant lineage. This suggests that the backbone of the chloroplast protein-import machinery evolved from the bacterial TamB–BamA protein-secretion system.
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We thank Y.-S. Teng for technical assistance at the initial stage of this project, M. Akita for the Cyanidioschyzon merolae 10D genomic DNA, the proteomics and imaging cores of the Institute of Molecular Biology and the proteomics core of the Institute of Biological Chemistry of Academia Sinica for technical assistance, ABRC for the tic236-1 and tic236-2 mutant seeds and the plant genome project of RIKEN Genomic Sciences Center for tic236-3 mutant seeds. This work was supported by the Ministry of Science and Technology (MOST 107-2321-B-001-001) and Academia Sinica of Taiwan (to H.-m.L.).
Nature thanks N. Pfanner, D. Schnell and S. Theg for their contribution to the peer review of this work.