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

摘要

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 (FtsH^trap) carrying a single amino acid exchange in the proteolytic center was expressed and purified in E. coli. FtsH^trap 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 FtsH^trap and wild-type FtsH (FtsH^WT) were purified, separated by two-dimensional PAGE, and subjected to mass spectrometry. Over-representation of LpxC in the FtsH^trap 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 FtsH^trap revealed previously unknown biological functions of the physiologically most important AAA⁺ protease in E. coli.