The role of the membrane-spanning domain of type I signal peptidases in substrate cleavage site selection

Type I signal peptidase (SPase I) catalyzes the cleavage of the amino-termi nal signal sequences from preproteins destined for cell export. Preproteins contain a signal sequence with a positively charged n-region, a hydrophobi c h-region, and a neutral but polar c-region, Despite having no distinct co nsensus sequence other than a commonly found c-region "Ala-X-Ala" motif pre ceding the cleavage site, signal sequences are recognized by SPase I with h igh fidelity, Remarkably, other potential Ala-X-Ala sites are not cleaved w ithin the preprotein. One hypothesis is that the source of this fidelity is due to the anchoring of both the SPase I enzyme (by way of its transmembra ne segment) and the preprotein substrate (by the h-region in the signal seq uence) in the membrane, This limits the enzyme-substrate interactions such that cleavage occurs at only one site, In this work we have, for the first time, successfully isolated Bacillus subtilis type I signal peptidase (SipS ) and a truncated version lacking the transmembrane domain (SipS-P2), With purified full-length as well. as truncated constructs of both B. subtilis a nd Escherichia coil (Lep) SPase I, in vitro specificity studies indicate th at the transmembrane domains of either enzyme are not important determinant s of in vitro cleavage fidelity, since enzyme constructs lacking them revea l no alternate site processing of pro-OmpA nuclease A substrate. In additio n, experiments with mutant pro-OmpA nuclease A substrate constructs indicat e that the h-region of the signal peptide is also not critical for substrat e specificity. In contrast, certain mutants in the c-region of the signal p eptide result in alternate site cleavage by both Lep and SipS enzymes.