Assembly of the Chloroplast ATP-Dependent Clp Protease in Arabidopsis Is Regulated by the ClpT Accessory Proteins  

Abstract

The ATP-dependent caseinolytic protease (Clp) is an essential housekeeping enzyme in plant chloroplasts. It is by far the most complex of all known Clp proteases, with a proteolytic core consisting of multiple catalytic ClpP and noncatalytic ClpR subunits. It also includes a unique form of Clp protein of unknown function designated ClpT, two of which exist in the model species Arabidopsis thaliana. Inactivation of ClpT1 or ClpT2 significantly reduces the amount of Clp proteolytic core, whereas loss of both proves seedling lethal under autotrophic conditions. During assembly of the Clp proteolytic core, ClpT1 first binds to the P-ring (consisting of ClpP3-6 subunits) followed by ClpT2, and only then does the P-ring combine with the R-ring (ClpP1, ClpR1-4 subunits). Most of the ClpT proteins in chloroplasts exist in vivo as homodimers, which then apparently monomerize prior to association with the P-ring. Despite their relative abundance, however, the availability of both ClpT proteins is rate limiting for the core assembly, with the addition of recombinant ClpT1 and ClpT2 increasing core content up to fourfold. Overall, ClpT appears to regulate the assembly of the chloroplast Clp protease, revealing a new and sophisticated control mechanism on the activity of this vital protease in plants.

Chloroplast Proteases: Updates on Proteolysis within and across Suborganellar Compartments

“…Clp core assembly and stabilization require plant-specific accessory proteins ClpT1/2 (Peltier et al, 2004;Sjögren and Clarke, 2011;Clarke, 2012;Kim et al, 2015). Lossof-function mutants for the ClpC1 chaperone and the ClpPRT core show pale-green, seedling-lethal, or embryodefective phenotypes, whereas knockouts for two adaptor proteins and ClpC2/D display no visible effects, underscoring their distinct contributions to plant development.…”

Conditional Depletion of the Chlamydomonas Chloroplast ClpP Protease Activates Nuclear Genes Involved in Autophagy and Plastid Protein Quality Control

“…However, the plastid ClpP protease also possesses unique accessory subunits (ClpT proteins) that are not found in Escherichia coli. Recent biochemical studies suggest that the ClpT subunits have evolved in order to regulate the assembly of the catalytic core (Sjögren and Clarke, 2011;Derrien et al, 2012).…”

Modified Clp Protease Complex in the ClpP3 Null Mutant and Consequences for Chloroplast Development and Function in Arabidopsis

“…Attached to the ClpPR core are ClpT1 and ClpT2, which have similarity to the N-terminal domain of the ClpC/ ClpD chaperones (Peltier et al, 2004;Olinares et al, 2011a). These ClpT subunits are unique to chloroplasts and have been hypothesized to regulate chaperone binding and/or substrate selection (Peltier et al, 2004;Olinares et al, 2011b) or to aid in the assembly of the ClpPR core complex (Sjögren and Clarke, 2011).…”

“…This showed that the ClpPR core consisted of one heptameric ring containing ClpP3, ClpP4, ClpP5, and ClpP6 in a 1:2:3:1 ratio (designated the P-ring) and the other ring containing ClpP1 and ClpR1, ClpR2, ClpR3, and ClpR4 in a 3:1:1:1:1 ratio (designated the R-ring). Moreover, based on biochemical and phylogenetic analysis, it was suggested that ClpT1 and ClpT2 bind to the adaxial side of the P-ring (Olinares et al, 2011a;Sjögren and Clarke, 2011). This Clp core complexity is puzzling and is very different from the much simpler Clp composition in photosynthetic and nonphotosynthetic bacteria.…”