Structural and functional analysis of adaptor proteins of the plant proteolytic Clp system

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

Puerto Madryn

Congreso; XLVI Reunión Anual SAIB; 2010

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

In chloroplasts, proteome homeostasis is regulated by a network of proteases,  molecular  chaperones  and  regulatory  proteins.  In particular,  the  tetradecameric  Clp  proteolytic  complex,  in assistance  with  Hsp100  chaperones,  selects,  disaggregates  and unfolds  proteins,  which  can  then  be  re-folded  or  directed  to proteolytic degradation. Little is known about the function of the Clp proteasome. In Escherichia coli,  the protein ClpS associates with  the  bacterial  Clp  complex  and  modulates  its  proteolytic activity.  In Arabidopsis  thaliana,  three proteins  (ClpT1/2 and ClpS) were proposed to play this role. We characterized these proteins in vitro  and  studied  their  interaction with Hsp100  chaperones. All proteins were expressed in recombinant E. coli cells and purified to homogeneity.  Since  Clp  proteins  are  usually  oligomeric,  we analyzed their assembly status by size exclusion chromatography. ClpT1 is a monomeric protein in the absence or presence of 5 mM ATP. Circular dichroism spectroscopy indicates that the protein is properly folded and that its thermal stability is rather low, with an unfolding transition at 45 ºC. ClpT1 stimulates the ATPase activity of the Hsp100 proteins ClpC2 and ClpD. Our results represent the first  evidence  of  the  regulatory  function  of  ClpT1  on  Hsp100 chaperones  and  will  contribute  to  elucidate  the  poorly  known regulatory  system of  the Clp complex  from plants.