Studying Proteases and Protein Turnover in Arabidopsis Chloroplasts

Proteolysis is a key process for maintaining homeostasis in all living cells. The ability to degrade specific metabolic enzymes and regulatory proteins is essential for both cellular integrity and function. Equally important is the efficient removal of damaged or otherwise inactive polypeptides, especially during periods of developmental change or stress adaptation. Being one of the most metabolically active plant organelles, chloroplasts require various proteases to control overall protein quality. Much has been revealed about these chloroplast proteases over the last decade, and yet the identity of their native protein substrates remains elusive. In this chapter, we describe a variation upon a classic genetic approach to identify protease substrates based on the comparative protein degradation rates in wild-type and transgenic lines with impaired proteolytic activity. We have successfully used this approach with an in organello assay to identify numerous substrates for the stromal Clp protease from Arabidopsis thaliana , using both gene knockout mutants and antisense repression lines. In principle, the technique can be readily adapted for the study of other chloroplast proteases, and in other plant and algal species as the necessary genetic resources become available.