Molecular chaperones and proteases are integral components of a multilayered protein quality control (PQC) network in all living cells. They regulate and maintain essential aspects of protein biogenesis and proteostasis during normal cellular conditions as well as in response to environmental changes that cause increased protein-folding stress. As such proteolysis is a fundamental process that not only plays a crucial role in the removal of damaged proteins from the cell but also serves as the ultimate negative regulatory element in the controlled turnover of regulatory proteins. Interestingly, in some cases the function and specificity of these proteases can be regulated by adaptor proteins, which facilitate the delivery of specific substrates to their cognate protease. In E. coli the adaptor protein ClpS controls the AAA+ protease ClpAP-mediated turnover of proteins containing N-terminal primary destabilizing residues, termed N-degrons. This pathway is known as the N-end rule pathway. Importantly, two of these primary destabilising residues (i.e. Leu and Phe) can be attached to the N-terminus of proteins in a non-ribosomal manner by the enzyme leucyl/phenylalanyl-tRNA-protein transferase (L/F-TR).
Although we have recently identified several putative physiological N-end rule substrates in E. coli, currently, the molecular details and significance of their regulated turnover remains unknown. Here, we focus on the analysis of these putative N-end rule substrates, with the aim to elucidate the substrate specificity of LFTR, using a variety of different approaches such as, peptide library screening, in vitro degradation assays and LFTR activity assays. Progress to date will be presented.