title: The role of the membrane-spanning domain of type I signal peptidases in substrate cleavage site selection.
abstract: Type I signal peptidase (SPase I) catalyzes the cleavage of the amino-terminal signal sequences from preproteins destined for cell export. Preproteins contain a signal sequence with a positively charged n-region, a hydrophobic h-region, and a neutral but polar c-region. Despite having no distinct consensus sequence other than a commonly found c-region "Ala-X-Ala" motif preceding the cleavage site, signal sequences are recognized by SPase I with high fidelity. Remarkably, other potential Ala-X-Ala sites are not cleaved within 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 transmembrane segment) and the preprotein substrate (by the h-region in the signal sequence) 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 and Escherichia coli (Lep) SPase I, in vitro specificity studies indicate that the transmembrane domains of either enzyme are not important determinants of in vitro cleavage fidelity, since enzyme constructs lacking them reveal no alternate site processing of pro-OmpA nuclease A substrate. In addition, experiments with mutant pro-OmpA nuclease A substrate constructs indicate that the h-region of the signal peptide is also not critical for substrate specificity. In contrast, certain mutants in the c-region of the signal peptide result in alternate site cleavage by both Lep and SipS enzymes.
authors.affiliation: Department of Chemistry, Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Carlos
authors.forename: M
authors.initials: M
authors.lastname: Paetzel
authors.forename: G
authors.initials: G
authors.lastname: Brubaker
authors.forename: A
authors.initials: A
authors.lastname: Karla
authors.forename: C M
authors.initials: CM
authors.lastname: Ashwell
authors.forename: M O
authors.initials: MO
authors.lastname: Lively
authors.forename: G
authors.initials: G
authors.lastname: Cao
authors.forename: P
authors.initials: P
authors.lastname: Bullinger
authors.forename: R E
authors.initials: RE
authors.lastname: Dalbey
mesh_terms: D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D011498:Protein Precursors / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D018528:ATP-Binding Cassette Transporters; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; C461599:maltose transport system, E coli; C057184:outer membrane protein A precursor (E coli); D012697:Serine Endopeptidases; C026515:type I signal peptidase