Multistep assembly of chloroplast NADH dehydrogenase-like subcomplex A requires several nucleus-encoded proteins, including CRR41 and CRR42, in Arabidopsis.

Chloroplast NADH dehydrogenase-like complex (NDH) mediates photosystem I cyclic electron transport and chlororespiration in thylakoids. Recently, substantial progress has been made in understanding the structure of NDH, but our knowledge of its assembly has been limited. In this study, a series of interactive proteomic analyses identified several stroma-localized factors required for the assembly of a stroma-protruding arm of NDH (subcomplex A). In addition to further characterization of the previously identified CHLORORESPIRATORY REDUCTION1 (CRR1), CRR6, and CRR7, two novel stromal proteins, CRR41 and CRR42, were discovered. Arabidopsis thaliana mutants lacking these proteins are specifically defective in the accumulation of subcomplex A. A total of 10 mutants lacking subcomplex A, including crr27/cpn60β4, which is specifically defective in the folding of NdhH, and four mutants lacking NdhL-NdhO subunits, were extensively characterized. We propose a model for subcomplex A assembly: CRR41, NdhO, and native NdhH, as well as unknown factors, are first assembled to form an NDH subcomplex A assembly intermediate (NAI500). Subsequently, NdhJ, NdhM, NdhK, and NdhI are incorporated into NAI500 to form NAI400. CRR1, CRR6, and CRR42 are involved in this process. CRR7 is likely to be involved in the final step, in which the fully assembled NAI, including NdhN, is inserted into thylakoids.