A Trapping Approach Reveals Novel Substrates and Physiological Functions of the Essential Protease FtsH in Escherichia coli

• Published in: The Journal of biological chemistry Vol. 287; no. 51; pp. 42962 - 42971
• Main Authors: Westphal, Kai, Langklotz, Sina, Thomanek, Nikolas, Narberhaus, Franz
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 14.12.2012; American Society for Biochemistry and Molecular Biology
• Subjects: ATP-dependent Protease; ATP-Dependent Proteases - isolation & purification; ATP-Dependent Proteases - metabolism; Bacteria; Enzyme Turnover; Escherichia coli; Escherichia coli - cytology; Escherichia coli - enzymology; Escherichia coli - growth & development; Escherichia coli Proteins - isolation & purification; Escherichia coli Proteins - metabolism; Iron-Sulfur Protein; Lipopolysaccharide (LPS); Microbial Viability; Microbiology; Models, Biological; Osmotic Pressure; Oxygen - metabolism; Phenotype; Protein Stability; Proteolysis; Proteome - metabolism; Proteomics - methods; Reproducibility of Results; Substrate Specificity; Bacteria; Iron-Sulfur Protein; Escherichia coli; Enzyme Turnover; ATP-dependent Protease; Lipopolysaccharide (LPS)
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1074/jbc.M112.388470

Proteolysis is a universal strategy to rapidly adjust the amount of regulatory and metabolic proteins to cellular demand. FtsH is the only membrane-anchored and essential ATP-dependent protease in Escherichia coli. Among the known functions of FtsH are the control of the heat shock response by proteolysis of the transcription factor RpoH (σ32) and its essential role in lipopolysaccharide biosynthesis by degradation of the two key enzymes LpxC and KdtA. Here, we identified new FtsH substrates by using a proteomic-based substrate trapping approach. An FtsH variant (FtsHtrap) carrying a single amino acid exchange in the proteolytic center was expressed and purified in E. coli. FtsHtrap is devoid of its proteolytic activity but fully retains ATPase activity allowing for unfolding and translocation of substrates into the inactivated proteolytic chamber. Proteins associated with FtsHtrap and wild-type FtsH (FtsHWT) were purified, separated by two-dimensional PAGE, and subjected to mass spectrometry. Over-representation of LpxC in the FtsHtrap preparation validated the trapping strategy. Four novel FtsH substrates were identified. The sulfur delivery protein IscS and the d-amino acid dehydrogenase DadA were degraded under all tested conditions. The formate dehydrogenase subunit FdoH and the yet uncharacterized YfgM protein were subject to growth condition-dependent regulated proteolysis. Several lines of evidence suggest that YfgM serves as negative regulator of the RcsB-dependent stress response pathway, which must be degraded under stress conditions. The proteins captured by FtsHtrap revealed previously unknown biological functions of the physiologically most important AAA+ protease in E. coli. Background: Only few substrates of the essential membrane-anchored protease FtsH are known. Results: New cytoplasmic and membrane-bound substrates of FtsH were trapped in vivo. Conclusion: FtsH is involved in the sulfatation of molecules, d-amino acid metabolism, and adaptation to anaerobiosis and stress conditions. Significance: The novel FtsH substrates significantly expand our knowledge on the biological functions of this fundamentally important protease.