CHARACTERIZATION OF THE CLPB/HSP70 THERMOTOLERANCE SYSTEM OF Arabidopsis thaliana CHLOROPLASTS

PARCERISA, IVANA L.; ROSANO, GERMÁN L.; CECCARELLI, EDUARDO A.

Rosario

Congreso; L Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2014

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Cells exposed to heat stress suffer perturbation of their protein homeostasis. Under these conditions, misfolded proteins lose their function and trigger aggregation of other proteins, threatening cell viability. To cope with these potential mishaps, cells have several defense mechanisms which include protein rescuing by molecular chaperones. In plants, an ATP-driven bi-chaperone system, ClpB/Hsp70, is thought to solubilize and reactivate aggregated proteins. The exact mechanism by which ClpB and Hsp70 act upon protein aggregates to promote their dissolution remains elusive and is completely unknown in plant chloroplasts. In this regard, we aim to unravel this mechanism and its role in the plant thermotolerance process. The chloroplastic Arabidopsis thaliana genes CLPB3, CPHSP70-1 and CPHSP70-2 were cloned and heterologously expressed as fusion proteins in suitable strains of Escherichia coli. All three proteins were successfully expressed and purified by IMAC. They were properly folded and displayed ATPase activity. Under our experimental conditions cpHsp70s exist in monomeric and dimeric forms as judged by gel filtration chromatography. cpHsp70-1 and -2 displayed different substrate preferences to E. coli extract proteins. Our working model is that they select the substrates that ClpB will subsequently disaggregate.