The chloroplast ClpP complex in Chlamydomonas reinhardtii contains an unusual high molecular mass subunit with a large apical domain

The composition of the chloroplast-localized protease complex, ClpP, from the green alga Chlamydomonas reinhardtii was characterized by nondenaturing electrophoresis, immunoblotting and MS. The detected ClpP complex has a native mass of approximate to 540 kDa, which is approximate to 200 kDa higher than ClpP complexes in higher plant chloroplasts, mitochondria or bacteria. The 540-kDa ClpP complex contains two nuclear-encoded ClpP proteins (ClpP3 and P5) and five ClpR (R1, R2, R3, R4 and R6) proteins, as well two proteins, ClpP1(L) and ClpP1(H), both probably derived from the plastid clpP1 gene. ClpP1(H) is 59 kDa and contains a approximate to 30-kDa insertion sequence (IS1) not found in other ClpP proteins, responsible for the high MW of the complex. Based on comparison with other sequences, IS1 protrudes as an additional domain on the apical surface of the ClpP/R complex, probably preventing interaction with the HSP100 chaperone. ClpP1(L) is a 25-kDa protein similar in size to other ClpP proteins and could arise by post-translational processing of ClpP1(H). Chloramphenicol-chase experiments show that ClpP1(L) and ClpP1(H) have a similar half-life, indicating that both are stable components of the complex. The structure of the ClpP complex is further discussed in conjunction with a phylogenetic analysis of the ClpP/R genes. A model is proposed for the evolution of the algal and plant complex from its cyanobacterial ancestor.