Molecular characterization of adaptor proteins ClpT1/2 and ClpS from Arabidopsis thaliana

COLOMBO, V.; ROSANO, G. L.; CECCARELLI, E. A.

Heidelberg

Simposio; EMBO / EMBL Simposia Quality Control – From Molecules to Organelles; 2012

EMBO / EMBL

In plant chloroplasts, proteolysis is an essential mechanism in the process of protein quality control. The chloroplastic ClpPR system of Arabidopsis thaliana is composed by ClpP1/3-6 and ClpR1-4, which self assemble into a proteolytic chamber. Substrate selection is thought to be carried out by the Hsp100 chaperones ClpC1/2 and ClpD. They bind proteins destined to degradation, unfold them and translocate them into the chamber. Three recently discovered proteins, ClpT1/2 and ClpS are thought to be adaptors and/or regulators of the Clp system. ClpT1/2 may assist in the assembly of the ClpPR complex and may regulate the binding of the Hsp100 chaperones to the core. ClpS is proposed to modulate ClpC1/2 substrate selection and affinity, presenting new targets to the system. ClpS from Escherichia coli is a recognin of the N-end rule degradation pathway. ClpS recognizes N-end rule substrates and presents them to ClpAP for degradation. Hence, ClpS of A. thaliana could have a similar function in chloroplasts. Nevertheless, there is little in vitro evidence to support these assumptions. We determined the oligomerization status of recombinant ClpT1 and ClpS by molecular exclusion chromatography and found that ClpT1 is monomeric and ClpS can form homo-oligomers of 2, 3, 4 and 6 subunits. We studied the interaction of these proteins with ClpC2 and ClpD by fast ultrafiltration analysis. The adaptor proteins were found to associate with the chaperones in the presence of ATP. Furthermore, we have investigated the influence of ClpT1 in the ATPase activity of ClpC2 and ClpD. ClpT1 has no influence in the activity of ClpC2; however, it stimulates ClpD ATPase activity by 60%, showing that ClpT1 is a specific activator of ClpD. Our results represent the first evidence of the regulatory function of ClpT1 in the Clp system and provide new insights for understanding regulation of proteostasis in chloroplasts.