THE CLPD AND CLPC1,2 CHAPERONES FOR THE CHLOROPLAST CLP PROTEASE SYSTEM PLAY UNIQUE ROLES IN DIRECTING PROTEOLYSIS
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To adapt to the dynamic demands of plant development and environmental conditions, the chloroplast proteome must undergo extensive remodeling by adjusting the balance of protein synthesis and degradation (proteostasis). The abundant CLP chaperone-protease system is essential for chloroplast proteostasis. CLP substrate selection is primarily determined by AAA+ chaperones, which unfold and directly translocate substrates into the protease core. The chloroplast CLP system in the plant model organism Arabidopsis thaliana has two homologs of the bacterial CLPC chaperone, CLPC1 and CLPC2, and a senescence-induced chaperone found only in land plants, CLPD. This thesis pursues a deeper understanding of the individual contributions of the three chloroplast CLP chaperones to maintaining proteostasis. In this thesis I examine the narrow, senescence-specific expression window of CLPD. I find that loss of CLPD expression accelerates Arabidopsis development while constitutive overexpression of CLPD delays development and is actively suppressed. By characterizing the in vivo CLPD interactome, I provide the first conclusive evidence that CLPD interacts with the CLPPR protease core as a true CLP chaperone. The CLPD interactome also identifies unique CLPD candidate substrates and suggests hetero-oligomerization of CLPD and CLPC. Based on these findings, I propose a role for CLPD in specifying CLP substrate turnover during senescence. Another distinction of CLPD is that it lacks the UVR dimerization motif found in CLPC1,2. Based on homology to bacterial CLPC proteins, I develop a model for how the ATPase and unfoldase activities of Arabidopsis CLPC1,2 may be regulated by UVR motifs through adaptor-dependent mechanisms. Additionally, I propose that the seven other Arabidopsis proteins with UVR motifs (UVR1-4, EX1,2, and CLPF) are recruited to the CLP system as substrates or adaptors by direct contacts between their UVR motifs and those of CLPC1,2. I determine that UVR2 and UVR3 are chloroplast-localized in vivo and likely form dimers capable of interacting with CLPC1 through UVR motif contacts. These efforts lay the groundwork for future investigation of the role of UVR motifs in chloroplast protein turnover by the CLP system.