MOLECULAR CHARACTERIZATION OF CHLOROPLASTIC Hsp100 CHAPERONES FROM Arabidopsis thaliana

ROSANO, G.L; CECCARELLI, E.A

Mar del Plata

Congreso; XLIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2007

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Molecular chaperones of the Hsp100 family have been identified in chloroplasts of Arabidopsis thaliana, namely ClpC1, ClpC2 and ClpD. By the use of knockout plants, it has been proposed that these proteins may have multiple roles such as protein folding assistance, protein disaggregation, proteolysis and precursor import into chloroplasts. The study of these activities is still in its infancy due to the fact that, to date, none of the chaperones have been purified. The main goals of this work were to purify them using a bacterial expression system and study their function in vitro. The cDNAs were obtained from the RIKEN A. thaliana cDNA bank, amplified by PCR and cloned into the vector pET28a. ClpC2 and ClpD were expressed and purified by affinity chromatography. They were recovered as properly folded dimers with expected molecular weight. The proteins have basal Mg2+-dependent ATPase activity. While both proteins have similar Km values, the Vmax value of ClpD was almost two-fold higher than that of ClpC2. The influence of temperature, pH, ionic strength and divalent cations was also assessed. Interestingly, ClpC2 was found to be the most heat stable member of the Clp family with a temperature optimum of 55 ºC. By contrast, ClpC1 could not be purified since its expression causes growth retardation and death to the host as judged by growth curves and viable cell counts.