The Bacillus subtilis ABC transporter EcsAB influences intramembrane proteolysis through RasP

Abstract

The Bacillus subtilis s W regulon is induced by different stresses that most probably affect integrity of the cell envelope. The activity of the extracytoplasmic function (ECF) sigma factor s W is modulated by the transmembrane anti-sigma factor RsiW, which undergoes stress-induced degradation in a process known as regulated intramembrane proteolysis, finally resulting in the release of s W and the transcription of s W -controlled genes. Mutations in the ecsA gene, which encodes an ATP binding cassette (ABC) of an ABC transporter of unknown function, block site-2 proteolysis of RsiW by the intramembrane cleaving protease RasP (YluC). In addition, degradation of the cell division protein FtsL, which represents a second RasP substrate, is blocked in an ecsA-negative strain. The defect in s W induction of an ecsA-knockout strain could be partly suppressed by overproducing RasP. A B. subtilis rasP-knockout strain displayed the same pleiotropic phenotype as an ecsA knockout, namely defects in processing a-amylase, in competence development, and in formation of multicellular structures known as biofilms. INTRODUCTIONGenes of Bacillus subtilis controlled by the alternative extracytoplasmic function (ECF) sigma factor s W constitute an antibiosis regulon (Helmann, 2002(Helmann, , 2006 that responds to a variety of envelope stresses such as certain antibiotics (Cao et al., 2002) and antimicrobial peptides (Pietiäinen et al., 2005;Butcher & Helmann, 2006), and also alkaline shock and phage infection . Its activity is modulated by RsiW, a transmembrane anti-sigma factor that sequesters and inactivates s W . Upon a stress signal, RsiW is degraded by the mechanism of regulated intramembrane proteolysis (RIP). In a concerted action, at least three proteases in three different compartments of the cell degrade the RsiW anti-sigma factor in a sequential manner, finally resulting in the release of s W and the transcription of s Wcontrolled genes. The first protease that has been identified to be involved in that process is RasP (regulating anti-sigma factor protease; formerly YluC). RasP belongs to the group of zinc-dependent intramembrane cleaving proteases (iClips) and cleaves RsiW in its transmembrane domain after the extracytoplasmic part of the anti-sigma factor has been removed by a site-1 protease (Schöbel et al., 2004). The site-2 degradation step catalysed by RasP uncovers a cryptic proteolytic tag that ensures further degradation of the RsiW remnant by cytoplasmic proteases, mainly ClpXP (Zellmeier et al., 2006). These two steps are related to RIP of the s E anti-sigma factor RseA of Escherichia coli (Ades, 2004;Alba & Gross, 2004). RasP is an orthologue of E. coli RseP, and the concept of a cryptic proteolytic tag recognized by ClpXP was first described for that system (Akiyama et al., 2004;Flynn et al., 2004). However, there is a marked difference in the site-1 proteolytic step, because no dependency on B. subtilis Deg (Htr) proteases was found and the inducing stress is apparently different. The probable site-1 prot...

Post-liberation cleavage of signal peptides is catalyzed by the site-2 protease (S2P) in bacteria

“…Clearance of SPs from the membrane might be important for protein secretion in this organism, as the B. subtilis rasP disruptant exhibits a weak defect in protein secretion (19). In accordance with our conclusion, it was reported that several peptide sex pheromones in Enterococcus faecalis are produced from the lipoprotein SP through S2P (Eep)-dependent, endoproteolytic processing (20,21).…”

Membrane Proteases in the Bacterial Protein Secretion and Quality Control Pathway

“…RseP was furthermore shown to cleave a ␤-lactamase signal peptide fused to the maltose binding protein in E. coli (2) and also the signal peptides of several prelipoproteins in Enterococcus faecalis and S. aureus, to generate sex pheromones (6,41). An SPP function of the S2P RasP would be consistent with the secretion defects described for a rasP mutant of B. subtilis (54), as signal peptides have a known inhibitory effect on preprotein translocation across the membrane (25,46,165). Processing of substrates by the prokaryotic S2P RseP and eukaryotic S2Ps is facilitated by helix destabilization at the site of cleavage (2,195).…”

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“…2C). The intramembrane-cleaving protease RasP has recently been shown to activate biofilm formation by B. subtilis (23). RasP is one of a number of proteases that is required to degrade RsiW, the W anti-sigma factor.…”