title: The N-degradome of Escherichia coli: limited proteolysis in vivo generates a large pool of proteins bearing N-degrons.
abstract: The N-end rule is a conserved mechanism found in Gram-negative bacteria and eukaryotes for marking proteins to be degraded by ATP-dependent proteases. Specific N-terminal amino acids (N-degrons) are sufficient to target a protein to the degradation machinery. In Escherichia coli, the adaptor ClpS binds an N-degron and delivers the protein to ClpAP for degradation. As ClpS recognizes N-terminal Phe, Trp, Tyr, and Leu, which are not found at the N terminus of proteins translated and processed by the canonical pathway, proteins must be post-translationally modified to expose an N-degron. One modification is catalyzed by Aat, an enzyme that adds leucine or phenylalanine to proteins with N-terminal lysine or arginine; however, such proteins are also not generated by the canonical protein synthesis pathway. Thus, the mechanisms producing N-degrons in proteins and the frequency of their occurrence largely remain a mystery. To address these issues, we used a ClpS affinity column to isolate interacting proteins from E. coli cell lysates under non-denaturing conditions. We identified more than 100 proteins that differentially bound to a column charged with wild-type ClpS and eluted with a peptide bearing an N-degron. Thirty-two of 37 determined N-terminal peptides had N-degrons. Most of the proteins were N-terminally truncated by endoproteases or exopeptidases, and many were further modified by Aat. The identities of the proteins point to possible physiological roles for the N-end rule in cell division, translation, transcription, and DNA replication and reveal widespread proteolytic processing of cellular proteins to generate N-end rule substrates.
authors: From the Laboratory of Cell Biology and.
authors: Matthew A
authors: MA
authors: Humbard
authors: Serhiy
authors: S
authors: Surkov
authors: Gian Marco
authors: GM
authors: De Donatis
authors: Lisa M
authors: LM
authors: Jenkins
authors: Michael R
authors: MR
authors: Maurizi
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000302:isolation & purification / Q000378:metabolism*; D055767:Immobilized Proteins / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020539:Sequence Analysis, Protein
chemical_list: D000596:Amino Acids; D029968:Escherichia coli Proteins; D055767:Immobilized Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins
keywords: ATP-dependent Protease; Aat; Adaptor Proteins; ClpA; ClpS; END Site; N-end Rule; Protein Degradation; Protein Processing; Proteomics
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