Stromal DUF760-1 and DUF760-2 proteins are degraded by the chloroplast Clp protease system but have very different half-lives

Abstract

The chloroplast AAA+ chaperone CLPC1 aids to select, unfold and deliver hundreds of proteins to the stromal CLP protease core complex for degradation. Through in vivo CLPC1 trapping we previously identified dozens of trapped proteins that are substrate adaptors (e.g. CLPS1 and CLPF), other chaperones (CLPC2 and CLPD) or (potential) substrates (e.g. RH3) for the CLP chaperone-protease system. Here we show that two of these highly trapped proteins, DUF760-1 and DUF760-2, are substrates for the chloroplast CLP protease system. In planta BiFC and yeast-2-hybrid analyses show that both DUF760 proteins can directly interact with the N-domain of CLPC1. Immunoblotting and confocal microscopy analysis demonstrates that both DUF760 proteins are highly enriched in clpc1-1 and clpr2-1 loss-of function mutants. In vivo cycloheximide chase assays in different genetic backgrounds show that DUF760-1 and 2 are both degraded by the chloroplast CLP protease. The half-life of DUF760-1 is 4-6 hours, whereas DUF760-2 is so unstable that it is very hard to detect unless degradation is inhibited in CLP loss-of-function alleles. Null mutants for DUF760-1 and DUF760-2 show weak but differential pigment phenotypes and differential sensitivity of protein translation inhibitors. The functions of these DUF760 proteins are unknown; the lack of shared mRNA co-expressors and the large difference in half-life and protein abundance, suggest that they play different roles within the chloroplast. Taken together, our results demonstrate that DUF760-1 and 2 are newly discovered substrates of CLP chaperone-protease system.