Competition co-immunoprecipitation reveals interactors of the chloroplast CPN60 chaperonin machinery

Abstract

The functionality of essential metabolic processes in chloroplasts depends on a balanced integration of nuclear-and chloroplast-encoded polypeptides into the plastids proteome. The chloroplast chaperonin machinery is an essential player in chloroplast protein folding with a more intricate structure and subunit composition compared to the orthologous GroEL/ES chaperonin of Escherichia coli. However, its exact role in chloroplasts remains obscure, mainly because of a very limited knowledge about the folded substrates. We employed the competition immunoprecipitation method for the identification of the chaperonins substrates in Chlamydomonas reinhardtii. Co-immunoprecipitation of the target complex in the presence of increasing amounts of isotope-labelled competitor epitope and subsequent mass spectrometry analysis specifically allowed to distinguish true interactors from unspecifically co-precipitated proteins. Besides known substrates such as RbcL, we revealed numerous new substrates with high confidence. Identified substrate proteins differ from bulk chloroplast proteins by a higher content of beta-sheets, lower alpha-helical content and increased aggregation propensity. Immunoprecipitations performed with a subunit of the co-chaperonin lid revealed the ClpP protease as a specific partner complex, with altered interactions during heat stress, pointing to a close collaboration of these machineries to maintain protein homeostasis in the chloroplast.