ClpS1 Is a Conserved Substrate Selector for the Chloroplast Clp Protease System in Arabidopsis

Abstract

ORCID ID: 0000-0001-9536-0487 (K.J.vW.).Whereas the plastid caseinolytic peptidase (Clp) P protease system is essential for plant development, substrates and substrate selection mechanisms are unknown. Bacterial ClpS is involved in N-degron substrate selection and delivery to the ClpAP protease. Through phylogenetic analysis, we show that all angiosperms contain ClpS1 and some species also contain ClpS1-like protein(s). In silico analysis suggests that ClpS1 is the functional homolog of bacterial ClpS. We show that Arabidopsis thaliana ClpS1 interacts with plastid ClpC1,2 chaperones. The Arabidopsis ClpS1 null mutant (clps1) lacks a visible phenotype, and no genetic interactions with ClpC/D chaperone or ClpPR core mutants were observed. However, clps1, but not clpc1-1, has increased sensitivity to the translational elongation inhibitor chloramphenicol suggesting a link between translational capacity and ClpS1. Moreover, ClpS1 was upregulated in clpc1-1, and quantitative proteomics of clps1, clpc1, and clps1 clpc1 showed specific molecular phenotypes attributed to loss of ClpC1 or ClpS1. In particular, clps1 showed alteration of the tetrapyrrole pathway. Affinity purification identified eight candidate ClpS1 substrates, including plastid DNA repair proteins and Glu tRNA reductase, which is a control point for tetrapyrrole synthesis. ClpS1 interaction with five substrates strictly depended on two conserved ClpS1 residues involved in N-degron recognition. ClpS1 function, substrates, and substrate recognition mechanisms are discussed.

Discovery of a Unique Clp Component, ClpF, in Chloroplasts: A Proposed Binary ClpF-ClpS1 Adaptor Complex Functions in Substrate Recognition and Delivery

“…In wild-type stroma, but only at very low levels in clpf-1 stroma, ClpS1 and ClpC1/2 were also detected in complexes between 200 and 600 kD (fractions 4 and 5) where there was no significant amount of ClpF. This indicates that ClpS1 can also interact with ClpC1/2 independent of ClpF (in agreement with previous in vitro experiments ;Nishimura et al, 2013) but that ClpF enhances interactions between ClpS1 and the ClpC chaperones, with ClpF possibly recruiting ClpS1 to ClpC.…”

“…RT-PCR and immunoblot analyses showed a complete loss of CLPF transcript and ClpF protein ( Figure 2C). The clpf plants were visibly indistinguishable from the wild type, which was similar to clps1 ( Figure 2B) but distinct from the pale-green or embryo lethal phenotypes of ClpPR core, clpc1, or clpt1 clpt2 double mutants Nishimura et al, 2013). We then generated a clpf-1 clps1 double null mutant, which did not show a visible phenotype either ( Figure 2B).…”

“…The MEP pathway enzyme 4-hydroxy-3-methylbutyl diphosphate synthase (HDS) was found to be strongly upregulated (2-to 10-fold) in clpc1, clpt1 clpt2 double mutants, and ClpPR mutants Zybailov et al, 2009;Nishimura et al, 2013). Here, we evaluated the quantitative response of the MEP pathway in clpf-1 compared with the wild type and clps1 ( Figure 3D).…”

MET1 Is a Thylakoid-Associated TPR Protein Involved in Photosystem II Supercomplex Formation and Repair in Arabidopsis

“…Peptide extracts for each gel band were then analyzed by online nanoliquid chromatography-MS/MS using an LTQ-Orbitrap (Thermo). Resulting spectral data were searched against the predicted Arabidopsis proteome (TAIR10), including a small set of typical contaminants and the decoy, as described (Nishimura et al, 2013). Only proteins with two or more matched spectra were considered.…”

Phosphorylation of GENOMES UNCOUPLED 4 Alters Stimulation of Mg Chelatase Activity in Angiosperms

“…These synchronized processes are under spatio-temporal control and are likely organized by means of auxiliary and assembly factors (Reisinger et al, 2008;Tanaka et al, 2010;Komenda and Sobotka, 2016). The first regulatory proteins of TBS have been shown to control metabolic flux through the pathway by modulating protein stability, enzyme activation, and inactivation (Meskauskiene et al, 2001;Tanaka et al, 2010;Czarnecki et al, 2011;Czarnecki and Grimm, 2012;Nishimura et al, 2013;Apitz et al, 2016).…”