Modification of PATase by L/F-transferase generates a ClpS-dependent N-end rule substrate in Escherichia coli

Abstract

The N-end rule pathway is conserved from bacteria to man and determines the half-life of a protein based on its N-terminal amino acid. In Escherichia coli, model substrates bearing an N-degron are recognised by ClpS and degraded by ClpAP in an ATP-dependent manner. Here, we report the isolation of 23 ClpS-interacting proteins from E. coli. Our data show that at least one of these interacting proteins-putrescine aminotransferase (PATase)-is posttranslationally modified to generate a primary N-degron. Remarkably, the N-terminal modification of PATase is generated by a new specificity of leucyl/phenylalanyltRNA-protein transferase (LFTR), in which various combinations of primary destabilising residues (Leu and Phe) are attached to the N-terminal Met. This modification (of PATase), by LFTR, is essential not only for its recognition by ClpS, but also determines the stability of the protein in vivo. Thus, the N-end rule pathway, through the ClpAPS-mediated turnover of PATase may have an important function in putrescine homeostasis. In addition, we have identified a new element within the N-degron, which is required for substrate delivery to ClpA.

A Single ClpS Monomer Is Sufficient to Direct the Activity of the ClpA Hexamer

“…Direct measurement of the effects of ClpS on degradation of non-N-end rule substrates is consistent with this model (46,47). Although substrates specifically regulated by ClpS-mediated degradation via the N-end rule have recently been identified (9,11), endogenous substrates that are primarily targeted by ClpAP are yet unknown.…”

“…In E. coli, the N-degrons include phenylalanine, tyrosine, tryptophan, and leucine (6). Arginine and lysine serve as secondary destabilizing residues, because proteins with these residues at the N terminus are modified by Leu/Phe-tRNA protein transferase to generate an N-degron by post-translational attachment of phenylalanine or leucine (8,9). Destabilizing residues are usually not found at the N terminus of newly synthesized proteins in vivo, because protein biosynthesis usually begins with methionine, and the N-terminal methionine is not removed by methionine aminopeptidase when one of these residues is in the second position (10).…”

“…Initially, components of the N-end rule pathway in E. coli were identified using proteins engineered to display N-degrons (6,8). Recently, however, endogenous substrates degraded by the N-end rule have been identified in E. coli (9,11). In at least one case, an aminopeptidase generates a protein with an N-degron that is degraded by ClpAP (9,11,12), but the mechanisms by which N-end rule substrates are formed and the frequency with which they occur are largely unknown.…”

Unfolded protein responses in bacteria and mitochondria: A central role for the ClpXP machine

“…1), by interacting with both the substrate and the AAA1 protease (8,(18)(19)(20). In these cases, a second element within the substrate may be required for its engagement with the translocation pore of the AAA1 protease (21).…”

The N-degradome of Escherichia coli

“…Two confirmed substrates of the E. coli system are DNA protection during starvation (Dps) and putrescine aminotransferase (PATase) (20,28). The N-degron for Dps, Leu at position 6 (28), is part of the primary amino acid sequence, and thus Dps is an Aat-independent substrate; how the truncated form of Dps with Leu 6 at the N terminus is generated is not known. PATase is a novel Aat-dependent substrate; its modification has two unique features.…”