Consequences of the loss of catalytic triads in chloroplast CLPPR protease core complexes in vivo

Abstract

The essential chloroplast CLP protease system consists of a tetradecameric proteolytic core with catalytic P (P1, 3–6) and non‐catalytic R (R1–4) subunits, CLP chaperones and adaptors. The chloroplast CLP complex has a total of ten catalytic sites,but it is not known how many of these catalytic sites can be inactivated before plants lose viability. Here we show that CLPP3 and the catalytically inactive variant CLPP3S164A fully complement the developmental arrest of the clpp3‐1 null mutant, even under environmental stress. In contrast, whereas the inactive variant CLPP5S193A assembled into the CLP core, it cannot rescue the embryo lethal phenotype of the clpp5‐1 null mutant. This shows that CLPP3 makes a unique structural contribution but its catalytic site is dispensable, whereas the catalytic activity of CLPP5 is essential. Mass spectrometry of affinity‐purified CLP cores of the complemented lines showed highly enriched CLP cores. Other chloroplast proteins were co‐purified with the CLP cores and are candidate substrates. A strong overlap of co‐purified proteins between the CLP core complexes with active and inactive subunits indicates that CLP cores with reduced number of catalytic sites do not over‐accumulate substrates, suggesting that the bottle‐neck for degradation is likely substrate recognition and unfolding by CLP adaptors and chaperones, upstream of the CLP core.

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

“…A recent study showed that there is a tight correlation between amino acid substitution rates in the plastid-encoded ClpP1 and the nuclear-encoded Clp subunits across a broad sampling of angiosperms, suggesting continuing selection on interactions within this complex ( 44 ). This is also consistent with the finding that ClpP5 (3 copies per Clp core) is crucial for Clp catalysis, whereas ClpP3 (one copy per core) plays an essential role in Clp structure even if its catalytic activity is dispensable ( 45 ). The ClpC1,2 and ClpD chaperones carry two ATPase domains and an IGF motif that is essential for binding to the Clp protease core complex.…”

“…Those two systems did not function well as a TRAP compared with the ClpC1 TRAP. No substrate candidates were identified, suggesting that the bottle neck for degradation is likely substrate recognition and unfolding by Clp adaptors and chaperones, upstream of the Clp core (45). study which showed that ClpB3 is critical for plastid biogenesis and possibly heat stress tolerance (50).…”

Extreme variation in rates of evolution in the plastid Clp protease complex

“…Recently, inactivation of the catalytic site in Arabidopsis thaliana ClpP3 was found to have no phenotypic effect, whereas the complete loss of ClpP3 resulted in a severe phenotype. In contrast, in the case of ClpP5, the loss of the catalytic site or the complete loss of the gene results in embryo lethality (Kim et al ., ; Liao et al ., ).…”

The Intron Retention Variant CsClpP3m Is Involved in Leaf Chlorosis in Some Tea Cultivars

“…It is known that the ClpP3 null mutation leads to seedling lethality in Arabidopsis (Kim et al, 2013). Point mutation of the serine-histidine-aspartic acid triad of ClpP3 shows that the loss of its catalytic core does not result in any visible changes in plant growth and development (Liao et al, 2018). However, possible effects of truncated CsClpP3 (CsClpP3m) lacking not only the catalytic core but also 178 amino acid residues, on leaf chlorosis needed to be further examined heterologously in tobacco plants since tea plants are difficult to genetically transform, which rules out endogenous gene expression through conventional Agrobacterium-mediated transformation approach or transient expression through infiltration.…”