Assembly of the chloroplast ATP-dependent Clp protease in Arabidopsis is regulated by the ClpT accessory proteins.

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.