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

Abstract

A TPases A ssociated with diverse cellular A ctivities (AAA+) are a superfamily of proteins that typically assemble into hexameric rings. These proteins contain AAA+ domains with two canonical motifs (Walker A and B) that bind and hydrolyze ATP, allowing them to perform a wide variety of different functions. For example, AAA+ proteins play a prominent role in cellular proteostasis by controlling biogenesis, folding, trafficking, and degradation of proteins present within the cell. Several central proteolytic systems ( e.g. , Clp, Deg, FtsH, Lon, 26S proteasome) use AAA+ domains or AAA+ proteins to unfold protein substrates (using energy from ATP hydrolysis) to make them accessible for degradation. This allows AAA+ protease systems to degrade aggregates and large proteins, as well as smaller proteins, and feed them as linearized molecules into a protease chamber. This review provides an up-to-date and a comparative overview of the essential Clp AAA+ protease systems in Cyanobacteria ( e.g ., Synechocystis spp), plastids of photosynthetic eukaryotes ( e.g. , Arabidopsis , Chlamydomonas ), and apicoplasts in the nonphotosynthetic apicomplexan pathogen Plasmodium falciparum . Recent progress and breakthroughs in identifying Clp protease structures, substrates, substrate adaptors ( e.g ., NblA/B, ClpS, ClpF), and degrons are highlighted. We comment on the physiological importance of Clp activity, including plastid biogenesis, proteostasis, the chloroplast Protein Unfolding Response, and metabolism, across these diverse lineages. Outstanding questions as well as research opportunities and priorities to better understand the essential role of Clp systems in cellular proteostasis are discussed.

Proteomics, phylogenetics, and coexpression analyses indicate novel interactions in the plastid CLP chaperone-protease system

“…This lack of identification of CLPS1 by MSMS is because it is a small protein (12 kDa) with relatively few suitable tryptic peptides (see also http://www.peptideatlas.org/builds/arabidopsis/); immunoblotting with CLPS1 specific serum previously showed that CLPS1 was enriched to the same extent as CLPF (11). All chloroplast CLPP (P1, 3,4,5,6), CLPR (R1,2,3,4) core subunits as well as the peripheral CLPT1,2 core proteins (1,2) were at least 2-fold enriched in CLPC1-TRAP as compared to CpC1-WT, whereas J o u r n a l P r e -p r o o f 7 CLPF, CLPC2 and CLPD were 4 to 7-fold enriched (Fig. 1B).…”

“…Forward and/or reverse genetics in Arabidopsis, maize, rice and tobacco demonstrated the essential nature of the plastid CLP system. Complete loss of CLPC chaperone or CLPPR protease capacity results in embryo lethality, whereas partial loss results in delayed growth and development, and virescent leaves (5)(6)(7). The plastid CLP system in Arabidopsis consists of a hetero-oligomeric protease core comprising one or more copies of five proteolytically active subunits (CLPP1 and CLPP3-6), four proteolytically inactive proteins (CLPR1-4), as well as two plant-specific accessory proteins (CLPT1,2), three AAA+ chaperones (CLPC1, CLPC2, CLPD), and two adaptors CLPS1 and CLPF.…”

Chlamydomonas proteases: classification, phylogeny, and molecular mechanisms

“…2A ), and many are related to the proteasomal and/or organellar protein quality control pathways ( Table S1 at Zenodo). Among them are the evolutionarily conserved Filamentation temperature-sensitive H (FtsH), Caseinolytic protease proteolytic subunit (ClpP), DegP, and Lon proteases, which are localized in the chloroplast and/or the mitochondrion and are well known for their role in maintaining organelle homeostasis ( Schuhmann and Adamska, 2012 ; Pinti et al , 2016 ; Kato and Sakamoto, 2018 ; Bouchnak and van Wijk, 2021 ). They digest misfolded proteins and protein aggregates induced by environmental stresses and thus have loose substrate cleavage preference, making substrate specificity screening a challenging task.…”

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.…”