STRUCTURAL AND FUNCTIONAL ANALYSES OF HSP100 MOLECULAR CHAPERONES FROM VASCULAR PLANTS AND BACTERIA

ROSANO, GERMÁN L.; BRUCH, EDUARDO M.; COLOMBO, CLARA V.; CECCARELLI, EDUARDO A.

Carlos Paz, Córdoba, Argentina

Congreso; XLIV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2008

Sociedad Argentina de Investigación Bioquímica

Molecular chaperones of the Hsp100 family have been identified in all kingdoms of life. Our work focuses on the Hsp100 family from chloroplasts of Arabidopsis thaliana (ClpC1, ClpC2 and ClpD or atHsp100s), ClpA from Escherichia coli and their proteolytic and regulatory partners. It has been proposed that these proteins may have roles in protein folding assistance, disaggregation, proteolysis and precursor import into chloroplasts. While some of these activities have been confirmed for ClpA, little is known about its chloroplastic counterparts mainly because they have not been purified. By means of affinity and gel filtration chromatography we were able to recover the atHsp100s as properly folded dimers with their expected molecular weight. They have basal Mg2+-dependent ATPase activity. The influence of temperature, pH, ionic strength and divalent cations on ATPase activity was also assessed. We also analysed the oligomerization status of the atHsp100s and their interaction with molecular partners (ClpS1). We then explored the functional homology between ClpA and the atHsp100s. The latter were expressed in an E. coli ClpA deficient strain and the resulting phenotype was studied using the green fluorescent protein as a probe for protein aggregation and degradation. Our results represent the first step to elucidate the poorly known protein quality control system in chloroplasts.