PROBING THE SPECIFICITY OF THE CHLOROPLASTIC N-RECOGNIN ATCLPS1 WITH FLUORESCENT PROBES BEARING DIFFERENT N-TERMINAL ENDS

LIC. ALEJO CANTOIA; DR. EDUARDO CECCARELLI; DR. GERMÁN ROSANO

Ciudad Autónoma de Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biocioencias 2017; 2017

Proteolysis is a highly regulated process as useful proteins must not be inadvertently eliminated. One of the mechanisms of target selection for degradation is recognition of N-degrons by an N-recognin. An N-degron is structural feature of the N-terminal end which is recognized by proteins (N-recognins) that deliver the target to a protease o mark it for degradation. In bacteria, the N-recognin ClpS recognizes destabilizing amino acids in the N-terminus, like Phe, Tyr, Trp and Leu, binds to the substrate and presents it to the ClpAP protease. Binding of ClpS is also enhanced by positive charges in second position; proteins starting with Phe-Arg (FR) are degraded within seconds. In chloroplasts of Arabidopsis thaliana, a homolog of bacterial ClpS has been found (AtClpS1) so it is believed that proteolysis of chloroplastic proteins is similar, although experimental evidence is lacking. Moreover, previous results by our group have shown that the specificity of AtClpS1 is different from bacterial ClpS as positive charges in second position seem to be detrimental for recognition. We created fluorescent probes to survey the specificity of AtClpS1, as it was done for bacterial ClpS. The N-terminal end of the green fluorescent protein was mutated to FR/FE/EE/EL and SA, being the last two N-terminal ends from two proposed natural substrates. The fluorescent probes were obtained in recombinant form and purified to homogeneity. By pull down assays and fluorescence anisotropy experiments, we show that AtClpS1 can bind to substrates with negative charges in the first and second position. This is the first experimental evidence of an N-recognin of the ClpS family with predilection for negative charges at or near the N-terminal end. Our results will help elucidating the sequence determinants of substrate recognition for degradation of chloroplastic proteins.