The chloroplast stromal CLP protease system is essential for growth and development. It consists of a proteolytic CLP core complex that likely dynamically interacts with oligomeric rings of CLPC1, CLPC2, or CLPD AAA+ chaperones. These ATP-dependent chaperones are predicted to bind and unfold CLP protease substrates, frequently aided by adaptors (recognins), and feed them into the proteolytic CLP core for degradation. To identify new substrates and possibly also new adaptors for the chloroplast CLP protease system, we generated an in vivo CLPC1 substrate trap with a C-terminal STREPII affinity tag in Arabidopsis thaliana by mutating critical glutamate residues (E374A and E718A) in the two Walker B domains of CLPC1 required for the hydrolysis of ATP (CLPC1-TRAP). On the basis of homology to nonplant CLPB/C chaperones, it is predicted that interacting substrates are unable to be released; that is, they are trapped. When expressed in the wild type, this CLPC1-TRAP induced a dominant visible phenotype, whereas no viable mutants that express CLPC1-TRAP in the clpc1-1 null mutant could be recovered. Affinity purification of the CLPC1-TRAP resulted in a dozen proteins highly enriched compared with affinity-purified CLPC1 with a C-terminal STREPII affinity tag (CLPC1-WT). These enriched proteins likely represent CLP protease substrates or new adaptors. Several of these trapped proteins overaccumulated in clp mutants or were found as interactors for the adaptor CLPS1, supporting their functional relationship to CLP function. Importantly, the affinity purification of this CLPC1-TRAP also showed high enrichment of all CLPP, CLPR, and CLPT subunits, indicating the stabilization of the CLPC to CLP core interaction and providing direct support for their physical and functional interaction.

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The essential chloroplast CLP protease system consists of a tetradecameric proteolytic core with catalytic P (P1, 3–6) and non‐catalytic R (R1–4) subunits, CLP chaperones and adaptors. The chloroplast CLP complex has a total of ten catalytic sites,but it is not known how many of these catalytic sites can be inactivated before plants lose viability. Here we show that CLPP3 and the catalytically inactive variant CLPP3S164A fully complement the developmental arrest of the clpp3‐1 null mutant, even under environmental stress. In contrast, whereas the inactive variant CLPP5S193A assembled into the CLP core, it cannot rescue the embryo lethal phenotype of the clpp5‐1 null mutant. This shows that CLPP3 makes a unique structural contribution but its catalytic site is dispensable, whereas the catalytic activity of CLPP5 is essential. Mass spectrometry of affinity‐purified CLP cores of the complemented lines showed highly enriched CLP cores. Other chloroplast proteins were co‐purified with the CLP cores and are candidate substrates. A strong overlap of co‐purified proteins between the CLP core complexes with active and inactive subunits indicates that CLP cores with reduced number of catalytic sites do not over‐accumulate substrates, suggesting that the bottle‐neck for degradation is likely substrate recognition and unfolding by CLP adaptors and chaperones, upstream of the CLP core.

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