Structural and functional analyses of hsp100 molecular chaperones from vascular plants and bacteria

ROSANO, G. L.; BRUCH, E. M.; COLOMBO, V.; CECCARELLI, E. A.

Villa Carlos Paz, Córdoca, Argentina

Congreso; XLIV Reunión Anual de la Sociedad Argentina de investigación en Bioquímica y Biología Molecular (SAIB); 2008

Sociedad Argentina de investigación en Bioquímica y Biología Molecular (SAIB)

Molecular chaperones of the Hsp100 family have been identified inall kingdoms of life. Our work focuses on the Hsp100 family fromchloroplasts of Arabidopsis thaliana (ClpC1, ClpC2 and ClpD oratHsp100s), ClpA from Escherichia coli and their proteolytic andregulatory partners. It has been proposed that these proteins mayhave roles in protein folding assistance, disaggregation, proteolysisand precursor import into chloroplasts. While some of theseactivities have been confirmed for ClpA, little is known about itschloroplastic counterparts mainly because they have not beenpurified. By means of affinity and gel filtration chromatography wewere able to recover the atHsp100s as properly folded dimers withtheir expected molecular weight. They have basal Mg2+-dependentATPase activity. The influence of temperature, pH, ionic strengthand divalent cations on ATPase activity was also assessed. We alsoanalysed the oligomerization status of the atHsp100s and theirinteraction with molecular partners (ClpS1). We then explored thefunctional homology between ClpA and atHsp100s. The latter wereexpressed in an E. coli ClpA deficient strain and the resultingphenotype was studied using the green fluorescent protein as a probefor protein aggregation and degradation. Our results represent thefirst step to elucidate the poorly known protein quality controlsystem in chloroplasts.