THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 265. No. 6, Issue of February 25, pp. 3417-3423, 1990 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S. A. Maturation of Escherichia coli Maltose-binding Protein by Signal Peptidase I in Viuo SEQUENCE REQUIREMENTS FOR EFFICIENT PROCESSING AND DEMONSTRATION OF AN ALTERNATE CLEAVAGE SITE* (Receivedfor publication, July 27, 1989) John D. Fikes$, Genevieve A. Barkocy-Gallagher, David G. Klapper, and Philip J. Bassford, Jr.5 From the Department of Microbiology and Immunology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7290 Comparative analyses of a number of secretory pro- serves to initiate export of the protein and which is subse- teins processed by eukaryotic and prokaryotic signal quently removed during or immediately following transloca- peptidases have identified a strongly conserved feature tion (1, 2). Most signal peptides share common structural regarding the residues positioned -3 and -1 relative features: a hydrophilic amino-terminal segment with l-3 basic to the cleavage site. These 2 residues of the signal residues followed by a 9-15-residue core of hydrophobic and peptide are thought to constitute a recognition site for neutral amino acids and finally a more polar carboxyl termi- the processing enzyme and are usually amino acids nus that immediately precedes the cleavage site (for review, with small, neutral side chains. It was shown previ- see 3). According to the loop model (4), the positively charged ously that the substitution of aspartic acid for alanine amino terminus interacts with the membrane surface, and the at -3 of the Escherichia coli maltose-binding protein (MBP) signal peptide blocked maturation by signal hydrophobic core inserts into and spans the lipid bilayer as a peptidase I but had no noticeable effect or MBP trans- loop or reverse hairpin structure, exposing the cleavage site location across the cytoplasmic membrane of its biolog- on the external face. Many details of this model remain a ical activity. This identified an excellent system in matter of conjecture; still, the experimental evidence supports which to undertake a detailed investigation of the this orientation of the signal peptide during translocation (1, structural requirements and limitations for the cleav- 5, 6). The enzymes responsible for the endoproteolytic re- age site. In vitro mutagenesis was used to generate 14 moval of the signal peptide are termed signal peptidases. different amino acid substitutions at -3 and 13 differ- Eukaryotic signal peptidase has been purified from several ent amino acid substitutions at -1 of the MBP signal tissue sources and is an integral membrane complex of two to peptide. The maturation of the mutant precursor spe- six polypeptides (7, 8). Two distinct signal peptidases have cies expressed in viuo was examined. Overall, the re- been purified from Escherichia coli cells (9). Both are integral sults obtained agreed fairly well with statistically de- membrane proteins composed of a single polypeptide. Signal rived models of signal peptidase I specificity, except peptidase II cleaves the signal peptides of lipoproteins exclu- that cysteine was found to permit efficient processing sively, and signal peptidase I processes all other secretory when present at either -3 and - 1, and threonine at - 1 proteins. resulted in inefficient processing. Interestingly, it was found that substitutions at -1 which blocked process- Although signal peptides exhibit little primary sequence ing at the normal cleavage site redirected processing, homology, the comparative analysis of numerous prokaryotic with varying efficiencies, to an alternate site in the and eukaryotic proteins has identified a strongly conserved signal peptide represented by the Ala-X-Ala sequence feature regarding the residues at positions -3 and -1 relative at positions -5 to -3. The substitution of aspartic acid to the cleavage site. It was noted that amino acids with small, for alanine at -5 blocked processing at this alternate neutral side chains predominated at these two positions (10, site but not the normal site. The amino acids occupying 11). von Heijne (12) compared 36 prokaryotic signal peptides the -5 and -3 positions in many other prokaryotic processed by signal peptidase I, and only alanine, glycine, signal peptides also have the potential for constituting leucine, serine, threonine, and valine were encountered at the alternate processing sites. This appears to represent -3 position. Somewhat more restrictive, position -1 was another example of redundant information contained found to harbor only alanine, glycine, serine, and threonine. within the signal peptide. Ala-X-Ala is the most frequently observed sequence preceding the cleavage site (10, 11). Based on these frequency analyses, positions -3 and -1 have been proposed to constitute a recognition site for the processing enzyme. These observa- Most secretory proteins are initially synthesized with an tions also were utilized to formulate the A-X-B model (10) amino-terminal extension designated the signal peptide which and the (-3,-l) rule (11) for predicting signal peptide cleav- * This research was supported by Grant A117292 from the National age sites. The (-3,-l) rule was later further modified by Institute of Allergy and Infectious Diseases. The costs of publication incorporating a larger statistical basis and a weighted matrix of this article were defrayed in part by the payment of page charges. approach (12). Various studies with E. coli have provided This article must therefore be hereby marked “aduertisement” in support for the basic tenets of these statistically based models accordance with 18 U.S.C. Section 1734 solely to indicate this fact. (6,13-15). However, such studies did not include a systematic $ Present address: Dept. of Biology 56-420,Massachusetts Institute of Technology, Cambridge, MA 02139. analysis of multiple substitutions at -3 or -1 to determine § To whom correspondence should be addressed. Tel.: 919-966- experimentally the requirements and limitations for efficient 1034. signal peptide processing. 3417 This is an Open Access article under the CC BY license. 3418 Processing of E. coli Maltose-binding Protein The MBP’ signal peptide is composed of 26 amino acid responding to either -3 (designated PS.2) or -1 (PS.7) of the signal residues and exhibits all the features typical of prokaryotic peptide are shown in Fig. 1. Each oligonucleotide was complementary to the DNA strand of packaged, single-stranded pJF8, and each signal peptides, including alanine residues at both -3 and -1 encoded the wild-type sequence except at the first 2 bases of the (16). The substitution of aspartic acid for alanine at -3 was codon to be mutagenized. The mutagenic oligonucleotides replaced found to block MBP processing by signal peptidase I without the amber mutation (TAG) at codon 23 of pJF8 with the wild-type having a noticeable effect on translocation across the cyto- sequence (TCG), allowing the identification of colonies harboring plasmic membrane (6). The preMBP remained anchored to mutagenized plasmids by their Mal+ phenotype on maltose tetrazo- the cytoplasmic membrane by its unprocessed signal peptide lium indicator agar supplemented with ampicillin. Plasmids from but was still capable of facilitating maltose uptake efficiently Mal+ colonies were reintroduced into cells of BAR1091 by phage M13-mediated transduction, and these strains were retained for sin- (6, 17). The characterization of this mutant MBP species gle-stranded plasmid DNA preparation and in uiuo analysis of MBP served to underscore the nonoverlapping nature of the hydro- export and processing. DNA sequence analysis performed as described phobic core and processing region and identified an excellent (24) was used to identify the l- or P-base substitutions present at the system to conduct mutational analysis of cleavage site struc- mutagenized codons. tural requirements with affecting protein translocation. In A 20-base mutagenic oligonucleotide coding for the ASP-~ alteration this study, oligonucleotide-directed mutagenesis has been uti- (designated malE22-1) was utilized to introduce this mutation into plasmids harboring various mutations at the codon for position -1 of lized to construct plasmids encoding an assortment of mutant the MBP signal peptide. Single-stranded DNA of each plasmid sub- preMBP species altered at specific positions near the signal stituted previously at codon -1 was mutagenized as described above, peptide processing site. The effect of 14 unique single amino except that mutagenized plasmids were identified solely by DNA acid substitutions at -3 and 13 unique substitutions at -1 on sequence analysis. Beginning with pJF2 harboring the malE22-1 processing were examined and the results compared with mutation, the oligonucleotide designated PS.ll (Fig. 1) was utilized predictions based on statistically derived models of signal to introduce various substitutions into the codon of the malE gene encoding the +2 residue. The mutagenesis reactions were performed peptidase I specificity. Interestingly, certain changes at -1 as described above. Specific mutagenic oligonucleotides (not shown) uncovered a nearby alternate cleavage site in the preMBP. also were synthesized to convert the Ala-’ codon of plasmid pJF13 to an aspartic acid and to delete codons -3 through -1 from plasmid MATERIALS AND METHODS pJF2. Radiolabeling of MBP, Immune Precipitation, SDS-PAGE, and Bacterial