Speaker
Description
Proteolysis is an essential process to maintain cellular homeostasis. One pathway that mediates selective protein degradation is the N-degron pathway, which relates the in vivo half-life of a protein to its N-terminal amino acid residue. In the cytosol of eukaryotes and prokaryotes, N terminal residues are major determinants of protein stability. While the eukaryotic N-degron pathway depends on the ubiquitin-proteasome-system, the prokaryotic one is driven by the Clp protease system. The adapter protein ClpS recognizes proteins bearing N-terminal destabilizing residues and delivers the substrate to the ClpAP chaperone-protease complex for degradation. Chloroplasts also contain the Clp protease system, including the adapter protein ClpS1, the chloroplast homologue of the bacterial substrate selector ClpS. Since chloroplasts are bacterially-derived, it seems reasonable to speculate that chloroplasts contain an N-degron pathway similar to the one in prokaryotes.
The aim of my PhD thesis is to investigate the impact of different N-terminal amino acids on chloroplast protein stability. Studying the stability of a protein dependent on its N-terminal residue requires a mechanism, which selectively exposes the amino acid at the N-terminus. In our case, a tobacco etch virus (TEV) protease based approach is used to activate the dormant N degron of a reporter protein. Plastid transformation in tobacco was used to generate plants with a plastid-encoded TEV protease. The reporter protein is introduced into the transformed plants using a transient transformation approach. Following translocation into the chloroplast, the plastid localized TEV protease can cleave the reporter protein and expose the desired N terminal residue. The outlined reporter system is used to, for the first time, systematically and comprehensively challenge the effect of specific N terminal amino acids on protein stability in this organelle.