Structural and Functional Analysis Of Adaptor Proteins Of The Plant Proteolytic Clp System

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

Puerto Madryn

Congreso; XLVI Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2010

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

In chloroplasts, proteome homeostasis is regulated by a network ofproteases, molecular chaperones and regulatory proteins. Inparticular, the tetradecameric Clp proteolytic complex, inassistance with Hsp100 chaperones, selects, disaggregates andunfolds proteins, which can then be re-folded or directed toproteolytic degradation. Little is known about the function of theClp proteasome. In Escherichia coli, the protein ClpS associateswith the bacterial Clp complex and modulates its proteolyticactivity. In Arabidopsis thaliana, three proteins (ClpT1/2 and ClpS)were proposed to play this role. We characterized these proteins invitro and studied their interaction with Hsp100 chaperones. Allproteins were expressed in recombinant E. coli cells and purified tohomogeneity. Since Clp proteins are usually oligomeric, weanalyzed their assembly status by size exclusion chromatography.ClpT1 is a monomeric protein in the absence or presence of 5 mMATP. Circular dichroism spectroscopy indicates that the protein isproperly folded and that its thermal stability is rather low, with anunfolding transition at 45 ºC. ClpT1 stimulates the ATPase activityof the Hsp100 proteins ClpC2 and ClpD. Our results represent thefirst evidence of the regulatory function of ClpT1 on Hsp100chaperones and will contribute to elucidate the poorly knownregulatory system of the Clp complex from plants.