Structural and Functional Insights into the Chloroplast ATP-Dependent CIp Protease in Arabidopsis.

In contrast with the model Escherichia coli CIp protease, the ATP-dependent CIp protease in higher plants has a remarkably diverse proteolytic core consisting of multiple CIpP and CIpR paralogs, presumably arranged within a dual heptameric ring structure. Using antisense lines for the nucleus-encoded CIpP subunit, CIpP6, we show that the Arabidopsis thaliana CIp protease is vital for chloroplast development and function. Repression of CIpP6 produced a proportional decrease in the CIp proteolytic core, causing a chlorotic phenotype in young leaves that lessened upon maturity. Structural analysis of the proteolytic core revealed two distinct subcomplexes that likely correspond to single heptameric rings, one containing the CIpP1 and cIpR1-4 proteins, the other containing CIpP3-6. Proteomic analysis revealed several stromal proteins more abundant in CIpP6 antisense lines, suggesting that some are substrates for the CIp protease. A proteolytic assay developed for intact chloroplasts identified potential substrates for the stromal CIp protease in higher plants, most of which were more abundant in young Arabidopsis leaves, consistent with the severity of the chlorotic phenotype observed in the cIpP6 antisense lines. The identified substrates all function in more general housekeeping roles such as plastid protein synthesis, folding, end quality control, rather than in metabolic activities such as photosynthesis. [ABSTRACT FROM AUTHOR]