An Unusual Signal Peptide Extension Inhibits the Binding of Bacterial Presecretory Proteins to the Signal Recognition Particle, Trigger Factor, and the SecYEG Complex

Janine H Peterson,Rose L Szabady,Harris D Bernstein

doi:10.1074/jbc.m508681200

Abstract

Considerable evidence indicates that the Escherichia coli signal recognition particle (SRP) selectively targets proteins that contain highly hydrophobic signal peptides to the SecYEG complex cotranslationally. Presecretory proteins that contain only moderately hydrophobic signal peptides typically interact with trigger factor (TF) and are targeted post-translationally. Here we describe a striking exception to this rule that has emerged from the analysis of an unusual 55-amino acid signal peptide associated with the E. coli autotransporter EspP. The EspP signal peptide consists of a C-terminal domain that resembles a classical signal peptide plus an N-terminal extension that is conserved in other autotransporter signal peptides. Although a previous study showed that proteins containing the C-terminal domain of the EspP signal peptide are targeted cotranslationally by SRP, we found that proteins containing the full-length signal peptide were targeted post-translationally via a novel TF-independent mechanism. Mutation of an invariant asparagine residue in the N-terminal extension, however, restored cotranslational targeting. Remarkably, proteins containing extremely hydrophobic derivatives of the EspP signal peptide were also targeted post-translationally. These and other results suggest that the N-terminal extension alters the accessibility of the signal peptide to SRP and TF and promotes post-translational export by reducing the efficiency of the interaction between the signal peptide and the SecYEG complex. Based on data, we propose that the N-terminal extension mediates an interaction with an unidentified cytoplasmic factor or induces the formation of an unusual signal peptide conformation prior to the onset of protein translocation.

Highlights

The vast majority of bacterial presecretory proteins contain a ϳ20 – 25-amino acid cleaved signal peptide that earmarks them for translocation across the inner membrane (IM)3 via a highly conserved heterotrimeric protein-conducting channel called the SecYEG complex
Proteins Containing EspPSP Are Targeted to the IM Post-translationally but Do Not Interact with trigger factor (TF)—In initial experiments, we sought to determine whether EspP is translocated across the IM by the SecYEG complex
In this report we show that a ϳ25 amino acid N-terminal extension associated with a long autotransporter signal peptide dramatically alters its targeting function

Summary

Function of an Unusual Signal Peptide Extension

The N-terminal 25 amino acids of the long autotransporter signal peptides contain a unique, highly conserved sequence motif(MNKIYX(Hy)X(Hy)X6(Hy)(V/I)(V/A)VSEL(A/S)R), where Hy is a large hydrophobic amino acid [22]. It is possible that the long signal peptides route autotransporters into the SRP pathway to ensure that they do not fold prematurely into a translocation-incompetent conformation in the cytoplasm. Several lines of evidence indicated that the signal peptide extension routed presecretory proteins into a post-translational targeting pathway by exerting an unprecedented inhibitory effect on their interaction with SRP, TF, and the SecYEG complex. To account for the data, we propose that the N-terminal extension of EspPSP either recruits a novel cytoplasmic factor that mediates a post-translational targeting reaction or causes the signal peptide to adopt an unusual conformation in which it has reduced affinity for the translocation machinery

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION