EARCHING FOR THE N-END RULE PATHWAY IN CHLOROPLASTS AND ITS SUBSTRATE SELECTOR

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

The caseinolytic protease (Clp) is crucial for chloroplast biogenesis and proteostasis. In Arabidopsis thaliana, Clp complexes are composed of the proteolytic chamber ClpPR and the chaperones ClpC 1/2 and ClpD, which select and unfold the substrates. The N-end rule degradation pathway states that the half-life of a protein is determined by the nature of its amino-terminal residue. Escherichia coli ClpS (ecClpS) recognizes N-end rule substrates and presents them to the ClpAP protease. Since ClpS is found in plants, we postulate the existence of the N-rule in chloroplasts. Gel filtration chromatography showed that the A. thaliana recombinant ClpS (atClpS) exists as dimers and trimers, in contrast with ecClpS, which is a monomer. These results were confirmed by chemical crosslinking. Also, atClpS was found to associate with Hsp 100 chaperones. We previously demonstrated that atClpS is able to bind substrates recognized by ecClpS. We designed mutant proteins in which the amino acids involved in substrate recognition were altered. We found that one amino acid of the recognition site is essential for peptide binding and that the surface charges at this site may define substrate specificity, which is different in plants and bacteria. Our results indicate that ClpS functions as a substrate selector of the Clp system and by the N-end pathway, discriminates a different spectrum of substrates.