Proteomics, phylogenetics, and co‐expression analyses indicate novel interactions in the plastid CLP chaperone‐protease system

Abstract

The chloroplast CLPC1 chaperone delivers and unfolds substrates to the stromal CLPPRT protease complex for degradation. We previously used an in vivo trapping approach to identify a dozen interactors with CLPC1 in Arabidopsis thaliana.This was achieved by expressing a STREPII‐tagged copy of CLPC1 mutated in its Walker B domains (CLPC1‐TRAP) followed by affinity purification and mass spectrometry. To screen for additional interactors, we carried out a far more sensitive and comprehensive in vivo protein trapping analysis. This identified 59 highly enriched CLPC1 protein interactors, in particular proteins in families of unknown functions (DUF760, DUF179, DUF3143, UVR‐DUF151, HugZ/DUF2470), as well as the UVR domain proteins EXE1 and EXE2 implicated in singlet oxygen damage and signaling. Phylogenetic and functional domain analyses identified other members of these families. These protein families appear to localize (nearly) exclusively to plastids. Several of these DUF proteins are of very low abundance as determined through the Arabidopsis PeptideAtlas http://www.peptideatlas.org/builds/arabidopsis/ showing that enrichment in the CLPC1‐TRAP was extremely selective. Evolutionary rate covariation indicated that the HugZ/DUF2470 family co‐evolved with the plastid CLP machinery suggesting functional and/or physical interactions. mRNA‐based co‐expression networks showed that all 12 CLP protease subunits tightly co‐expressed as a single cluster with deep connections to DUF760‐3. Co‐expression modules for other trapped proteins suggested specific functions in biological processes, e.g. UVR2 and UVR3 were associated with extra‐plastidic degradation, whereas DUF760‐6 is likely involved in senescence. This study provides a strong foundation for discovery of substrate selection by the chloroplast CLP protease system.