Dissecting The Molecular Process Of N-End Rule Substrate Recognition And Delivery To Chaperones By The Chloroplastic N-Recognin AtClpS1

AGUILAR LUCERO, DIANELA; ROSANO, GERMÁN LEANDRO; CECCARELLI, EDUARDO AUGUSTO; COLOMBO, CLARA VICTORIA

Buenos Aires

Congreso; Reunión conjunta de sociedades de Biociencias; 2017

Sociedad Argentina de Biología

Proteolysis is one of the main strategies for proteostasis maintenance. In chloroplasts of Arabidopsis thaliana, the caseinolytic protease (Clp) is the main proteolytic system. It is composed by the protease ClpPR (a complex of 9 different proteins), the Hsp100 unfoldases ClpC1/2 and AtClpS1, an N-recognin who mediate substrate recognition. N-recognins identify structural features in the N-terminal end of the target, a process known as the N-end rule. Bacterial ClpS is extensively characterized, mainly due to the possibility of reconstituting the bacterial Clp system in vitro and evaluating the degradation of fluorescent targets, a strategy that could not be replicated for the chloroplast system. To unravel the specificity of the plant proteolytic machinery and to exploit the usefulness of the in vitro activity of the bacterial system, we rationally designed five chimeric proteins using the AtClpS1 backbone, exchanging different pieces of its sequence with the corresponding part of bacterial ClpS. We focused specially on the parts that mediate binding to the bacterial chaperone with the ultimate goal of obtaining a fully functional bacterial system but with plant specificity. The five chimeras were successfully obtained in recombinant form and purified. Preliminary results show that the N-terminal end of AtClpS1 is essential for substrate delivery, as the chimera NtEcClpS::Nt-AtClpS1 greatly inhibited the degradation of fluorescent substrates. Our work provides new molecular tools to unravel the N-end rule in chloroplasts