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Abstract Different proteases and peptidases are present within chloroplasts and nonphotosynthetic plastids to process precursor proteins and to degrade cleaved chloroplast transit peptides and damaged, misfolded, or otherwise unwanted proteins. Collectively, these proteases and peptidases form a proteolysis network, with complementary activities and hierarchies, and build-in redundancies. Furthermore, this network is distributed across the different intra-chloroplast compartments (lumen, thylakoid, stroma, envelope). The challenge is to determine the contributions of each peptidase (system) to this network in chloroplasts and nonphotosynthetic plastids. This will require an understanding of substrate recognition mechanisms, degrons, substrate, and product size limitations, as well as the capacity and degradation kinetics of each protease. Multiple extra-plastidial degradation pathways complement these intra-chloroplast proteases. This review summarizes our current understanding of these intra-chloroplast proteases in Arabidopsis and crop plants with an emphasis on considerations for building a qualitative and quantitative network view. 

Abstract The chloroplast chaperone CLPC1 aids to select, unfold, and deliver hundreds of proteins to the CLP protease for degradation. Through in vivo CLPC1, trapping we previously identified dozens of proteins that are (potential) substrate adaptors or substrates for the CLP chaperone–protease system. In this study, we show that two of these highly trapped proteins, DUF760-1 and DUF760-2, are substrates for the CLP protease in Arabidopsis (Arabidopsis thaliana). Loss-of-function mutants and transgenic plants were created for phenotyping, protein expression, and localization using immunoblotting and confocal microscopy. In planta BiFC, cycloheximide chase assays, and yeast 2-hybrid analyses were conducted to determine protein interactions and protein half-life. Both DUF760 proteins directly interacted with the N-domain of CLPC1 and both were highly enriched in clpc1-1 and clpr2-1 mutants. Accordingly, in vivo cycloheximide chase assays demonstrated that both DUF760 proteins are degraded by the CLP protease. The half-life of DUF760-1 was 4 to 6 h, whereas DUF760-2 was highly unstable and difficult to detect unless CLP proteolysis was inhibited. Null mutants for DUF760-1 and DUF760-2 showed weak but differential pigment phenotypes and differential sensitivity to protein translation inhibitors. This study demonstrates that DUF760-1 and DUF760-2 are substrates of the CLP chaperone–protease system and excellent candidates for the determination of CLP substrate degrons. 

Abstract Proteolysis, including post-translational proteolytic processing as well as protein degradation and amino acid recycling, is an essential component of the growth and development of living organisms. In this article, experts in plant proteolysis pose and discuss compelling open questions in their areas of research. Topics covered include the role of proteolysis in the cell cycle, DNA damage response, mitochondrial function, the generation of N-terminal signals (degrons) that mark many proteins for degradation (N-terminal acetylation, the Arg/N-degron pathway, and the chloroplast N-degron pathway), developmental and metabolic signaling (photomorphogenesis, abscisic acid and strigolactone signaling, sugar metabolism, and postharvest regulation), plant responses to environmental signals (endoplasmic-reticulum-associated degradation, chloroplast-associated degradation, drought tolerance, and the growth-defense trade-off), and the functional diversification of peptidases. We hope these thought-provoking discussions help to stimulate further research.