Synthetic Signal Peptides Specifically Recognize SecA and Stimulate ATPase Activity in the Absence of Preprotein

Abstract

Although it is known that virtually all exported proteins require a signal peptide, it is not clearly understood how the signal peptide interfaces with the translocation machinery to achieve transport. In this study we document a direct interaction between the signal peptide and SecA, a primary component of the translocase in Escherichia coli, and show that the signal peptide itself can stimulate SecA-lipid ATPase activity. Using synthetic signal peptides corresponding to the wild type alkaline phosphatase signal sequence and two model sequences, we find that the extent of stimulation

Chloroplast SecA and Escherichia coli SecA Have Distinct Lipid and Signal Peptide Preferences

“…2C) with an optimum corresponding to an approximately 1,000:1 molar ratio of lipid to SecA. This is consistent with a requirement for SecA to be sufficiently ensconced in lipid to generate the fully active species (19). Flotation analysis (17) confirms that, under these conditions, cpSecA is associated with the membranes (Fig.…”

“…2A). That cpSecA activity peaks at 20% DOPG is interesting in view of the strong dependence of protein translocation on anionic lipids in vivo in E. coli (17,27) and the optimum of 40 to 60% observed for signal peptide-stimulated E. coli SecA-lipid ATPase activity in vitro (19). The requirement for 20% DOPG is, however, consistent with the content of anionic lipids found in the thylakoid membranes of higher plants (30).…”

“…In bacteria, the components of this pathway include the SecYEG complex, which constitutes the translocation channel (8, 11), and SecA, an ATPase that powers the translocation of the polypeptide across the cytoplasmic membrane (16,31,32). In Escherichia coli, the efficiency of protein translocation is directly proportional to the amount of anionic phospholipids (17,20,25) and this reflects the effect of these lipids on SecA ATPase activity (19,29). In plants, genes encoding chloroplast homologues of SecY (15) and SecE (10) have been identified, as has a SecA homologue from several chloroplast sources (22,24,26,28).…”

“…The effect of lipid composition on the ATPase activity of cpSecA was examined using established assays (17,19,29). Keeping the concentrations constant for cpSecA (40 g/ml) and lipid (400 g/ml), we investigated the cpSecA-lipid ATPase activity in lipid systems composed of dioleoylphosphatidylcholine (DOPC)-DOPG, dioleoylphosphatidylethanolamine (DOPE)-DOPG, or DGDG-DOPG.…”

Demonstration of a Specific Escherichia coli SecY−Signal Peptide Interaction

“…Consequently, it is tempting to consider whether the signal peptide interacts specifically with SecY as one might expect if it takes an active role in directing the preprotein through the export relay system. To address this issue we used purified His (6) -SecEYG (referred to as SecYEG hereafter) and a synthetic peptide corresponding to the wild-type alkaline phosphatase signal sequence which we have characterized extensively in vivo (38) and in vitro (39,40). As shown in Figure 1A, in the presence of SecYEG-reconstituted proteoliposomes, SecA ATPase activity increased about 3-fold compared to its endogenous activity.…”