In vivo proteolytic profiling of the type I and type II metacaspases in Chlamydomonas reinhardtii exposed to salt stress

Metacaspases are cysteine proteases present in plants, fungi and protists. While the association of metacaspases with cell death is studied in a range of organisms, their native substrates are largely unknown. Here, we explored the in vivo proteolytic landscape of the two metacaspases, CrMCA-I and CrMCA-II, present in the green freshwater alga Chlamydomonas reinhardtii, using N-terminal peptide labelling and enrichment during standard conditions and salt stress. Comparison between the cleavage events of CrMCA-I and CrMCA-II in metacaspase mutants revealed unique cleavage preferences and substrate specificity. Degradome analysis demonstrated relevance of the predicted metacaspase substrates to the physiology of Chlamydomonas reinhardtii cells and its adaptation during salt stress. Functional enrichment analysis indicated an involvement of CrMCA-I in photosynthesis and translation, while CrMCA-II seems to be important during the catabolism of carboxylic acids. Altogether, our findings suggest distinct cellular functions of the two metacaspases in C. reinhardtii during salt stress response.