Proteomics, phylogenetics, and coexpression analyses indicate novel interactions in the plastid CLP...

Area of Science:

• Plant Biology
• Molecular Biology
• Proteostasis

Background:

• The chloroplast chaperone CLPC1 facilitates substrate unfolding and delivery to the CLPPRT protease complex for degradation.
• Previous in vivo trapping identified CLPC1 interactors, but a more comprehensive analysis was needed to expand the pool of candidate substrates, adaptors, and regulators.

Purpose of the Study:

• To conduct a highly sensitive and comprehensive in vivo protein trapping analysis to identify a broader range of CLPC1 interactors in Arabidopsis thaliana.
• To characterize the identified interactors, including those with unknown functions (DUFs) and UVR domain proteins, and infer their potential roles in chloroplast proteostasis.

Main Methods:

• Utilized an in vivo protein trapping approach with a mutated CLPC1 (CLPC1-TRAP) in Arabidopsis thaliana.
• Employed affinity purification coupled with mass spectrometry to identify and enrich CLPC1 protein interactors.
• Performed phylogenetic analysis, functional domain analysis, and mRNA-based coexpression network analysis to characterize identified proteins and their interactions.

Main Results:

• Identified 59 highly enriched CLPC1 protein interactors, including DUF families (DUF760, DUF179, DUF3143, UVR-DUF151, HugZ/DUF2470) and UVR domain proteins (EXE1, EXE2).
• Phylogenetic and domain analyses suggested plastid localization for several DUF proteins, with some being of very low abundance, indicating high selectivity of the CLPC1-TRAP enrichment.
• Evolutionary rate covariation indicated coevolution of the HugZ/DUF2470 family with the CLP machinery, and coexpression networks linked specific DUF proteins to processes like senescence and degradation.

Conclusions:

• This study provides a robust foundation for understanding substrate selection mechanisms within the chloroplast CLP protease system.
• The identified interactors, particularly DUF and UVR domain proteins, represent novel candidates for involvement in chloroplast protein quality control and signaling pathways.
• The integration of coexpression and coevolutionary analyses offers insights into the functional specialization and coordination of the chloroplast proteolytic machinery.