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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-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 co...
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Published in: | The Journal of biological chemistry 2000-12, Vol.275 (49), p.38813-38822 |
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Main Authors: | , , , , , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | 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 âAla- X -Alaâ 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. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M007093200 |