In Vivo Trapping of Proteins Interacting with the Chloroplast CLPC1 Chaperone: Potential Substrates and Adaptors

Abstract

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.

Structure, function, and substrates of Clp AAA+ protease systems in cyanobacteria, plastids, and apicoplasts: A comparative analysis

“…2). It should be noted that the Clp chaperones may accumulate as dimers (e.g., in chloroplasts) when not engaged in the degradation cycle and that formation of the chaperone hexamer requires priming of the chaperone by adaptors and/or ATP, leading to the formation of the activate hexamer in the ATP-bound state (9,29,30).…”

“…Clp adaptors and chaperones must selectively recognize the degradation signal in substrates through their substrateinteracting domains and deliver the substrates to the proteolytic chamber (21). Confirmed and candidate Clp substrates have been identified in cyanobacteria, chloroplasts, and apicoplasts by (i) a combination of comparative proteomics and follow-up studies, as reviewed (20,21), (ii) discovery through direct interaction assays with adaptors ClpS1 and/or ClpF (43,61) or NblA (62), or (iii) in vivo (in planta) trapping with the plastid ClpC chaperone (29) or ClpP, ClpR, ClpC, and ClpS proteins in apicoplasts (53), or (iv) targeted analysis of specific proteins to probe for the role of Clp instability, for example, for UmuD in cyanobacteria (42).…”

“…An in vivo trapping approach in Arabidopsis identified a dozen potential substrates interacting with Arabidopsis chloroplast ClpC1 (29), following strategies successfully used for substrates trapping of other AAA+ proteins in bacterial systems, as reviewed (11). The in vivo trap was generated by expressing ClpC1 mutated in two critical glutamate residues in the two Walker B domains of ClpC1 required for the hydrolysis of ATP and with a C-terminal STREPII affinity tag for purification (29) (Fig.…”

Rapid sequence evolution is associated with genetic incompatibilities in the plastid Clp complex

“…Likewise, disruption of nuclear genes that encode core Clp subunits also produces severe phenotypic effects (Sjögren, et al 2006;Koussevitzky, et al 2007;Kim, et al 2009;Moreno, et al 2017). The Clp complex plays a central role in protein quality control and homeostasis in plastids, and many potential proteolytic targets have now been identified (Majeran, et al 2000;Nishimura, et al 2013;Tapken, et al 2015;Apitz, et al 2016;Pulido, et al 2016;Moreno, et al 2018;Welsch, et al 2018;Montandon, et al 2019).…”

Gene duplication and rate variation in the evolution of plastid ACCase and Clp genes in angiosperms

“…The Clp complex is one of the most abundant stromal proteases and degrades a variety of targets (Apitz et al, 2016;Bouchnak and van Wijk, 2021;Majeran et al, 2000;Montandon et al, 2019;Nishimura et al, 2017;Welsch et al, 2018). This complex consists of many types of subunits.…”