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Title: THE MEMBRANE SPANNING DOMAIN OF TYPE I SIGNAL PEPTIDASE DOES NOT PLAY A DIRECT ROLE IN CLEAVAGE SITE SELECTION

Author
item CARLOS, JOSEPH - OHIO STATE UNIVERSITY
item PAETZEL, MARK - UNIV. OF BRITISH COLUMBIA
item BRUBAKER, GREG - OHIO STATE UNIVERSITY
item KARLA, ANDREW - OHIO STATE UNIVERSITY
item Ashwell, Christopher
item LIVELY, MARK - WAKE FOREST UNIVERSITY
item GUOQING, CAO - OHIO STATE UNIVERSITY
item BULLINGER, PATRICK - OHIO STATE UNIVERSITY
item DALBEY, ROSS - OHIO STATE UNIVERSITY

Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/26/2000
Publication Date: N/A
Citation: N/A

Interpretive Summary: Type I signal peptidase (SPase I) catalyzes the cleavage of the amino- terminal signal sequences from pre-proteins destined for cell export. Pre- proteins contain a signal sequence with no distinct consensus sequence other than a commonly found c-region "Ala-X-Ala" motif preceding the cleavage site where signal sequences are recognized by SPase I with high fidelity. Remarkably, other potential "Ala-X-Ala" sites are not cleaved within the pre-protein. One hypothesis is that the source of this accuracy is due to anchoring 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). Studies indicate that the transmembrane domains of either enzyme are not important determinants of cleavage fidelity, as enzyme constructs lacking them reveal no alternate site processing of pro-OmpA nuclease A substrate (PONA). In addition, experiments with mutant PONA substrate constructs indicate that other regions of the signal peptide are also not critical for substrate specificity. In contrast, certain mutants in the c-region of the signal peptide result in alternate site cleavage by bacterial signal peptidases.

Technical Abstract: Type I signal peptidase (SPase I) catalyzes the cleavage of the amino- terminal signal sequences from pre-proteins destined for cell export. Pre- proteins 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 pre-protein. 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 pre-protein 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 B. 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 E. 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 as enzyme constructs lacking them reveal no alternate site processing of pro- OmpA nuclease A substrate (PONA). In addition, experiments with mutant PONA 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.