Identification and characterisation of physiological substrates of the N-end rule protein degradation pathway in Escherichia coli

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Biochemistry, La Trobe Institute for Molecular Science (LIMS), School of Molecular Sciences, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.

The N-end rule pathway is conserved from bacteria to humans and determines the half-life of a protein based on its N-terminal amino acid. In Escherichia coli, model substrates bearing a destabilising N-terminal amino acid (N-degron) are degraded by ClpAP in an ATP-dependent manner. The degradation of these substrates is controlled by the adaptor protein ClpS, which is directly responsible for recognition of the N-degron and its delivery to the ClpAP protease. Previous to this thesis there were no known substrates of the N-end rule pathway in prokaryotes. To investigate the N-end rule in bacteria a pull down approach using recombinant ClpS was developed and applied to a proteomic wide search for ClpS substrates. This thesis describes the identification of 23 ClpS interacting proteins isolated from E. coli. Two of these proteins, DNA protection during starvation protein (Dps) and putrescine aminotransferase (PATase), are post-translationally modified to generate an N-degron. In the first example, the N-degron of Dps appears to result from an endoproteolytic cleavage by an unknown protease. In contrast, the N-degron of PATase is generated by the addition of primary destabilising residues to the N-terminus of the protein by the enzyme leucyl/phenylalanyl-tRNA-protein transferase (LFTR). Thus LFTR determines the stability of the protein in vivo. Independent of the type of post-translational modification, both N-end rule substrates are degraded by ClpAP in a ClpS dependent manner. An investigation into other LFTR dependent substrates lead to the partial characterization of two other putative ClpAPS substrates, the beta subunit of the acetyl-CoA carboxylase (AccD) and aldehyde dehydrogenase (AldB), both of which contain N-degrons. The characterization of the first bacterial N-end rule substrates has provided insight into the role of the prokaryotic N-end rule pathway in E. coli, linking it to stress pathways and metabolic homeostasis.