The ATPase activity and assembly of ClpB3/Hsp100 from Arabidopsis thaliana chloroplasts

PARCERISA, I; ROSANO, G. L.; CECCARELLI, E. A.

Mar del Plata

Congreso; LI Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB).; 2015

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

To counteract protein aggregation, bacteria, fungi and plants contain a bi-chaperone system composed of ATP dependent Hsp70 and ClpB/Hsp100 chaperones, which rescue aggregated proteins and provide thermotolerance to cells. The ClpB component of this system is an AAA+ ATPase that forms an hexameric functional ring-like structure of identical protomers, each one containing two nucleotide binding domains (NBD-1 and -2). ClpB oligomerize in response to different stimuli such as nucleotide binding or ionic strength, which in turn regulate the activity of the chaperone. In this work, we provide the first characterization of the ClpB3/Hsp100 from A. thaliana chloroplasts. Oligomerization in the presence of ATP was studied by SEC and the innate ATPase activity analyzed under several conditions by the Malachite Green method. We have established that ATP stimulates hexamerization while high ionic strength favors disassembly of the complexes. Furthermore, we have determined the kinetic parameters of the enzymeand its activity under a range of pH values and temperatures. ClpB3 lacks activity at pH values equal to or lower than 4.5 and has an optimal activity at temperatures around 62oC. Since the overall behavior of the enzyme resembles that of bacterial and yeast homolog, these results suggest that ClpB3 from A. thaliana could act similarly in the acquisition of plant thermotolerance