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

AGUILAR LUCERO, DIANELA; COLOMBO, CLARA V; CECCARELLI, EDUARDO A; ROSANO, GERMÁN L

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de BioCiencias; 2017

Sociedades de BioCiencias

enance.In chloroplasts of Arabidopsis thaliana, the caseinolyticprotease (Clp) is the main proteolytic system. It is composed bythe protease ClpPR (a complex of 9 different proteins), the Hsp100unfoldases ClpC1/2 and AtClpS1, an N-recognin who mediate substraterecognition. N-recognins identify structural features in theN-terminal end of the target, a process known as the N-end rule.Bacterial ClpS is extensively characterized, mainly due to the possibilityof reconstituting the bacterial Clp system in vitro and evaluatingthe degradation of fluorescent targets, a strategy that could not bereplicated for the chloroplast system. To unravel the specificity ofthe plant proteolytic machinery and to exploit the usefulness of thein vitro activity of the bacterial system, we rationally designed fivechimeric proteins using the AtClpS1 backbone, exchanging differentpieces of its sequence with the corresponding part of bacterialClpS. We focused specially on the parts that mediate binding to thebacterial chaperone with the ultimate goal of obtaining a fully functionalbacterial system but with plant specificity. The five chimeraswere successfully obtained in recombinant form and purified. Preliminaryresults show that the N-terminal end of AtClpS1 is essentialfor substrate delivery, as the chimera NtEcClpS::Nt-AtClpS1 greatlyinhibited the degradation of fluorescent substrates. Yet in anotherapproach, we designed an AtClpS1 protein, where the predicted At-ClpS1 amino acids involved in Hsp100 interaction were mutated.Pull down experiments showed that these amino acids are crucialfor AtClpS1 and ClpC2 interaction. This mutant will be used to fishout substrates from the chloroplast stroma and sequence their N-terminalend. Our work provides new molecular tools to unravel theN-end rule in chloroplasts.