Bacillus Subtilis Intramembrane Protease RasP Activity in Escherichia Coli and

RasP is a predicted intramembrane metalloprotease of that has been proposed to cleave the stress response anti-sigma factors RsiW and RsiV, the cell division protein FtsL, and remnant signal peptides within their transmembrane segments. To provide evidence for direct effects of RasP on putative substrates, we developed a heterologous coexpression system. Since expression of catalytically inactive RasP E21A inhibited expression of other membrane proteins in , we added extra transmembrane segments to RasP E21A, which allowed accumulation of most other membrane proteins. A corresponding active version of RasP appeared to promiscuously cleave coexpressed membrane proteins, except those with a large periplasmic domain. However, stable cleavage products were not observed, even in mutant Fusions of transmembrane segment-containing parts of FtsL and RsiW to maltose-binding protein (MBP) also resulted in proteins that appeared to be RasP substrates upon coexpression in , including FtsL with a full-length C-terminal domain (suggesting that prior cleavage by a site 1 protease is unnecessary) and RsiW designed to mimic the PrsW site 1 cleavage product (suggesting that further trimming by extracytoplasmic protease is unnecessary). Purified RasP cleaved His-MBP-RsiW(73-118) within the RsiW transmembrane segment based on mass spectrometry analysis, demonstrating that RasP is an intramembrane protease. Surprisingly, purified RasP failed to cleave His-MBP-FtsL(23-117). We propose that the lack of α-helix-breaking residues in the FtsL transmembrane segment creates a requirement for the membrane environment and/or an additional protein(s) in order for RasP to cleave FtsL. Intramembrane proteases govern important signaling pathways in nearly all organisms. In bacteria, they function in stress responses, cell division, pathogenesis, and other processes. Their membrane-associated substrates are typically inferred from genetic studies in the native bacterium. Evidence for direct effects has come sometimes from coexpression of the enzyme and potential substrate in a heterologous host and rarely from biochemical reconstitution of cleavage We applied these two approaches to the enzyme RasP and its proposed substrates RsiW and FtsL. We discovered potential pitfalls and solutions in heterologous coexpression experiments in , providing evidence that both substrates are cleaved by RasP but, surprisingly, that only RsiW was cleaved , suggesting that FtsL has an additional requirement.