N-degron specificity of chloroplast ClpS1 in plants

The prokaryotic N-degron pathway depends on the Clp chaperone-protease system and the ClpS adaptor for recognition of N-degron bearing substrates. Plant chloroplasts contain a diversified Clp protease, including the ClpS homolog ClpS1. Several candidate ClpS1 substrates have been identified, but the N-degron specificity is unclear. Here, we employed in vitro ClpS1 affinity assays using eight N-degron green fluorescence protein reporters containing either F, Y, L, W, I, or R in the N-terminal position. This demonstrated that ClpS1 has a restricted N-degron specificity, recognizing proteins bearing an N-terminal F or W, only weakly recognizing L, but not recognizing Y or I. This affinity is dependent on two conserved residues in the ClpS1 binding pocket and is sensitive to FR dipeptide competition, suggesting a unique chloroplast N-degron pathway.