Inhibition of purified Escherichia coli leader peptidase by the leader (signal) peptide of bacteriophage M13 procoat

Abstract

The leader peptide of bacteriophage M13 procoat inhibited the cleavage of M13 procoat or pre-maltosebinding protein by purified Escherichia coli leader peptidase. This finding confirms inferences that the leader is the primary site of enzyme recognition and suggests a rationale for the rapid hydrolysis of leader peptides in vivo.Escherichia coli leader peptidase is an integral membrane protein of the plasma membrane (12) which cleaves presecretory and membrane proteins after they cross the membrane bilayer (1,14). The released leader peptide (prepeptide or signal peptide) is rapidly degraded. The specificity of leader peptidase is somewhat unusual in that it cleaves a wide variety of preproteins which do not share a consensus sequence. However, leader peptides do possess common structural features (7,9). Amino acid residues with small side chains are commonly found at positions -1 and -3 (with respect to the cleavage site), and a helix-breaking glycine or proline is found in residues -4 to -6. There is no apparent sequence conservation in the mature region of secreted proteins. Genetic studies have established that conserved residues of the leader are necessary for cleavage rather than for the process of membrane insertion (4, 5). Short peptides which span the cleavage site of bacteriophage M13 procoat, a membrane protein precursor (10), have been prepared by controlled proteolysis of the purified protein and were found to be accurately cleaved by leader peptidase (la). These studies suggested that leader peptidase only recognizes the leader peptide.To test this idea, we assayed the ability of chemically synthesized M13 procoat leader peptide to inhibit leader peptidase. This peptide has 23 residues from the initiator methionine to the alanine which immediately preceeds the procoat-cleavage site. The chemically synthesized M13 procoat leader peptide (purchased from Dennis Olshevsky, University of California, San Diego) was dissolved at 1 mg/ml in 0.05 M Tris chloride (pH 8.5)-0.1% (vol/vol) Triton X-100 (buffer A). Radiochemically pure 35S-procoat (8), purified leader peptidase (11), and leader peptide, each at the indicated concentrations in 20-1LI reaction mixtures, were incubated for 1 h at 37°C. Samples were then analyzed for cleavage by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography (3). While 10 ng of leader peptidase was sufficient to cleave 50% of the procoat to coat in our standard assay (Fig. 1A, lane 5), 10 ,ug was required for equivalent cleavage in the presence of 250 pg of leader peptide per ml, corresponding to a 1,000-fold inhibition of the enzyme. Titration of the leader peptide (Fig. 1B) showed inhibition at levels of 2 ,ug of leader peptide per ml. To confirm that the inhibition was indeed due to the leader * Corresponding author.peptide rather than to a contaminant in the synthesis reaction, the leader peptide was purified by high-pressure liquid chromatography. Fractions were collected across the region of the single peak of absorbance and were assayed for inhibit...

Signal Peptide Peptidase- and ClpP-like Proteins of Bacillus subtilis Required for Efficient Translocation and Processing of Secretory Proteins

“…First, these signal peptides could affect the activity of the SecA protein, which was previously shown to be inhibited in vitro by synthetic signal peptides (43). Second, the accumulation of signal peptides could affect the activity of the signal peptidases of B. subtilis, as it was previously shown that, like SecA, the signal peptidase of E. coli was also inhibited in vitro by synthetic signal peptides (44). Compared with the tepA mutation, the effects of the sppA disruption were rather mild, as they could only be shown under conditions of AmyQ hypersecretion.…”

The Bacillus subtilis ABC transporter EcsAB influences intramembrane proteolysis through RasP

“…Signal peptide peptidases described for eukaryotes belong to the group of intramembrane cleaving proteases, and attack certain signal peptides after they have been clipped from newly synthesized secretory or membrane proteins (Weihofen & Martoglio, 2003). One function of RasP might be to degrade certain signal peptides in the membrane, and in the absence of RasP activity the accumulation of these peptides might inhibit signal sequence processing by leader peptidases, as has been shown in vitro for synthetic signal peptides (Wickner et al, 1987). We have tried to discern (signal) peptides in isolated membranes of B. subtilis rasP-negative and other strains several times using different methods, but so far the methods have proved to be problematic.…”

Competition between functional signal peptides demonstrates variation in affinity for the secretion pathway

“…The integral membrane proteins SecE and SecY are thought to form a protein-conducting channel for the translocating polypeptide (1,6), and SecG stimulates the translocation activity (31). The leader peptidase is responsible for cleavage of the signal peptide (47), while SecD may be important in the release of the mature protein from the membrane (29). A second set of proteins involved in targeting exported proteins to the membrane includes the E. coli SRP (Ffh), a cytosolic ribonucleoprotein composed of 4.5S RNA and a single polypeptide homologous to the 54-kDa subunit of eukaryotic SRP, and FtsY, a GTPase homologous to the ␣ subunit of the SRP receptor on the endoplasmic reticulum (26).…”