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J Biol Chem. 1992 Jan 15;267(2):1231-8.

Synthesis of precursor maltose-binding protein with proline in the +1 position of the cleavage site interferes with the activity of Escherichia coli signal peptidase I in vivo.

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Department of Microbiology and Immunology, School of Medicine, University of North Carolina, Chapel Hill 27599-7290.


The residues occupying the -3 and -1 positions relative to the cleavage site of secretory precursor proteins are usually amino acids with small, neutral side chains that are thought to constitute the recognition site for the processing enzyme, signal peptidase. No restrictions have been established for residues positioned +1 to the cleavage site, although there have been several indications that mutant precursor proteins with a proline at +1 cannot be processed by Escherichia coli signal peptidase I (also called leader peptidase). A maltose-binding protein (MBP) species with proline at +1, designated MBP27-P, was translocated efficiently but not processed when expressed in E. coli cells. Unexpectedly, induced expression of MBP27-P was found to have an adverse effect on the processing kinetics of five different nonlipoprotein precursors analyzed, but not precursor Lpp (the major outer membrane lipoprotein) processed by a different enzyme, signal peptidase II. Cell growth also was inhibited following induction of MBP27-P synthesis. Substitutions in the MBP27-P signal peptide that blocked MBP translocation across the cytoplasmic membrane and, hence, access to the processing enzyme or that altered the signal peptidase I recognition site at position -1 restored both normal growth and processing of other precursors. Since overproduction of signal peptidase I also restored normal growth and processing to cells expressing unaltered MBP27-P, it was concluded that precursor MBP27-P interferes with the activity of the processing enzyme, probably by competing as a noncleavable substrate for the enzyme's active site. Thus, although signal peptidase I, like many other proteases, is unable to cleave an X-Pro bond, a proline at +1 does not prevent the enzyme from recognizing the normal processing site. When the RBP signal peptide was substituted for the MBP signal peptide of MBP27-P, the resultant hybrid protein was processed somewhat inefficiently at an alternate cleavage site and elicited a much reduced effect on cell growth and signal peptidase I activity. Although the MBP signal peptide also has an alternate cleavage site, the different properties of the RBP and MBP signal peptides with regard to the substitution of proline at +1 may be related to their respective secondary structures in the processing site region.

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