Modification of PATase by L/F-transferase generates a ClpS-dependent N-end rule substrate in Escherichia coli.
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 post-translationally modified to generate a primary N-degron. Remarkably, the N-terminal modification of PATase is generated by a new specificity of leucyl/phenylalanyl-tRNA-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.
Ninnis
Robert L
RL
Department of Biochemistry, La Trobe University, Melbourne, Victoria, Australia.
Spall
Sukhdeep K
SK
Talbo
Gert H
GH
Truscott
Kaye N
KN
Dougan
David A
DA
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002846:Chromatography, Affinity; D004151:Dipeptides / Q000378:metabolism; D049071:Endopeptidase Clp / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008024:Ligands; D053858:Metabolic Networks and Pathways; D008954:Models, Biological; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity; D000637:Transaminases / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; C454820:ClpS protein, E coli; D004151:Dipeptides; D029968:Escherichia coli Proteins; D008024:Ligands; D050505:Mutant Proteins; C088010:dps protein, E coli; D019881:Aminoacyltransferases; C021511:leucyltransferase; D000637:Transaminases; C483568:ClpA protease, E coli; C483572:ClpP protease, E coli; D049071:Endopeptidase Clp