UNRAVELING THE CHLOROPLASTIC N-END RULE: SEARCHING FOR SEQUENCE DETERMINANTS FOR PROTEIN DEGRADATION

CECCARELLI, EDUARDO A; ROSANO, GERMÁN L; AGUILAR LUCERO, DIANELA

Salta

Congreso; LV Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2019

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Proteins that are to be eliminated must be proficiently recognized by proteolytic systems so that inadvertent elimination of useful proteins isavoided. One mechanism to ensure proper recognition is the presence of N-terminal degradation signals (N-degrons) that are targeted by adaptorproteins (N-recognins). The members of the caseinolytic protease S (ClpS) family of N-recognins identify targets bearing an N-terminalphenylalanine, tyrosine, tryptophan or leucine residue, and then present them to a protease system. This process is known as the ?bacterial N-endrule?. The presence of a ClpS protein in Arabidopsis thaliana chloroplasts (ClpS1) prompted the hypothesis that the bacterial N-end rule exists inthis organelle. However, there is no experimental evidence on this and reports on the specificity of ClpS1 show conflicting data. In this work, weset up to unravel the specificity of ClpS1 by the use of peptide arrays, membrane-bound peptides synthesized directly on cellulose membranes.The arrays were ordered to the manufacturer specifying the sequence of the peptides attached to the membrane. Hundreds of different N-terminalsequences can be designed and probed simultaneously. The interactor protein (in our case, recombinant ClpS1) was incubated with the membrane,which is then thoroughly washed. Bound ClpS1 was then immunodetected by anti-ClpS1 antibodies. A colorimetric signal indicates stronginteraction to the peptide in that location, whose sequence is known. Bound peptides were also ordered in their N-terminal acetylated version toanalyze the effect of this post translational modification on ClpS binding. Our results show that ClpS1 can bind to peptides bearing the canonicalbacterial N-degrons (Phe, Tyr, Trp and Leu). Binding to peptides starting with Phe and Tyr was readily detected. However, signals indicatingbinding to Leu-bearing peptides were of much lower intensity, suggesting lower affinity. Also, a second array in which the second residue wasalways proline or serine and the first one was systematically surveyed showed that ClpS1 did not recognize Leu-Ser or Leu-Pro; N-degrons thatare readily recognized by bacterial ClpS proteins. Also, N-acetylation of the peptides in the membrane completely abolished the interaction withClpS1. This indicates that this posttranslational modification may greatly influence protein half-life in chloroplasts. Overall, it can be concludedthat ClpS1 form plant chloroplasts has a similar recognition pattern compared to bacterial ClpS but also presents differences that suggest that thechloroplastic N-end rule presents unique characteristics on its own.