Insights into the Hsp100-transit peptide recognition process

BRUCH, EDUARDO M.; ROSANO, GERMÁN L; CECCARELLI, EDUARDO A

Puerto Madryn, Argentina

Congreso; XLVI Reunión Anual SAIB; 2010

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Molecular chaperones of the Hsp100 family have been identified in all kingdoms of life. It has been proposed that these proteins play a key  role  in protein  folding assistance, disaggregation, proteolysis and precursor  import  into chloroplasts. Some of  these activities have been  previously  observed  investigating  an Hsp100  form  E.  coli called ClpA. However, the role of Hsp100 in chloroplast import has not  been  elucidated.  It  has  been  postulated  that  a  crucial  step  in protein translocation into plastids requires the binding of an Hsp100 to  an  amino-terminal  region  of  the  protein  called  transit  peptide (TP). However,  evidences  of Hsp100-TP  interaction  are  lacking. The goal of  this work  is  to demonstrate  this  interaction and gain further  insight  into  its nature, using  the TP of pea  ferredoxin NADP+ reductase  (FNR) as a model and  two Hsp100 chaperons  from A. thaliana  (  ClpC2  and  ClpD).  TP  fused  to  the  GST  N-terminus interacts with both plant  chaperones. On  the  contrary,  fusions  in which the TP was placed at the carboy-terminus of GST or between GST and FNR were not recognized by the chaperones. Moreover, evidences were  obtained  indicating  that  interaction with  the  full transit peptide was lower than a shorter version of this peptide. Our results show for the first time in vitro evidence for the interaction between a TP and an Hsp100  from A. Thaliana.