In the N‐degron pathway of protein degradation of Escherichia coli, the N‐recognin ClpS identifies substrates bearing N‐terminal phenylalanine, tyrosine, tryptophan, or leucine and delivers them to the caseinolytic protease (Clp). Chloroplasts contain the Clp system, but whether chloroplastic ClpS1 adheres to the same constraints is unknown. Moreover, the structural underpinnings of substrate recognition are not completely defined. We show that ClpS1 recognizes canonical residues of the E. coli N‐degron pathway. The residue in second position influences recognition (especially in N‐terminal ends starting with leucine). N‐terminal acetylation abrogates recognition. ClpF, a ClpS1‐interacting partner, does not alter its specificity. Substrate binding provokes local remodeling of residues in the substrate‐binding cavity of ClpS1. Our work strongly supports the existence of a chloroplastic N‐degron pathway.

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