We have reconstituted protein translocation across plasma membrane vesicles of Escherichia coli using purified proOmpA and trigger factor, a 63 kd soluble protein. Treatment of membrane vesicles with urea inactivates them for translocation unless a factor present in cytoplasmic extracts is added during the translocation reaction. Sedimentation analysis showed that the stimulatory activity is of distinctly higher mol. wt than trigger factor. Cytoplasmic extracts from a strain that greatly overproduces the SecA protein are highly enriched in the stimulatory activity for untreated membranes and restore translocation to urea‐treated membranes, suggesting that this protein is the stimulatory factor. This assay was used to monitor the isolation of SecA protein from the overproducing strain. The purified protein is soluble, yet binds peripherally to membranes with high affinity and supports translocation. Using pure proOmpA, SecA protein, trigger factor and urea‐treated membranes, the protein export process was resolved into binding and translocation steps. We find that proOmpA binds to membrane vesicles with or without SecA protein, but that translocation only occurs when SecA was bound prior to proOmpA.

The precursor protein proOmpA can translocate across purified Escherichia coli inner membrane vesicles in the absence of any other soluble proteins. ProOmpA, purified 2000‐fold in the presence of 8 M urea, is competent for translocation following rapid renaturation via dilution. ATP, the transmembrane electrochemical potential, and functional secY protein are essential for the translocation of proOmpA renatured by dilution. The kinetics of its translocation and the level of translocation at each concentration of ATP are indistinguishable from that of proOmpA renatured by dialysis with trigger factor. After dilution, the proOmpA rapidly loses its competence for membrane assembly. However, this competence is stabilized by trigger factor. Assembly‐competent proOmpA is in a protease‐sensitive conformation, whereas proOmpA which has lost this competence is more resistant to degradation. This suggests that the primary role for trigger factor in in vitro protein translocation is to maintain precursor proteins in a translocation‐competent conformation. We propose that a properly folded precursor protein and ATP are the only soluble components which are essential for bacterial protein translocation.

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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...

Down-regulation in vivo of liver plasma membrane receptors for prostaglandin E (PGE) was investigated in Sprague-Dawley rats using the 16,16-dimethyl analogue of PGE2, This analogue was used for subcutaneous injections because it escapes the rapid pulmonic degradation characteristic of PGE and was recognized well by liver plasma membrane receptors. Following treatment with the analogue, the concentration of PGE receptors was significantly decreased (-37%, P less than 0.001), but the binding affinity was not altered. There was no evidence for carry-through of the analogue into the isolated plasma membrane preparation. It was also demonstrated that GTP decreased the binding affinity between PGE and its receptor. Down-regulation of receptor concentration was associated with a significant decrease (P less than 0.001) in PGE1-stimulated plasma membrane adenylate cyclase activity. These data provide the novel demonstration that rat liver plasma membrane receptor for PGE can be down-regulated in vivo and that this causes a corresponding decrease in PGE-induced plasma membrane adenylate cyclase activity.