A trapping approach reveals novel substrates and physiological functions of the essential protease Ftsh in Escherichia coli

Abstract

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. AnFtsH variant (FtsH trap) 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 (FtsH WT) 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. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.