Abstract

Protein translocation across the bacterial cytoplasmic membrane is an essential process catalyzed by the Sec translocase, which in its minimal form consists of the protein-conducting channel SecYEG, and the motor ATPase SecA. SecA binds via its positively charged N-terminus to membranes containing anionic phospholipids, leading to a lipid-bound intermediate. This interaction induces a conformational change in SecA, resulting in a high-affinity association with SecYEG, which initiates protein translocation. Here, we examined the effect of anionic lipids on the SecA-SecYEG interaction in more detail, and discovered a second, yet unknown, anionic lipid-dependent event that stimulates protein translocation. Based on molecular dynamics simulations we identified an anionic lipid-enriched region in vicinity of the lateral gate of SecY. Here, the anionic lipid headgroup accesses the lateral gate, thereby stabilizing the pre-open state of the channel. The simulations suggest flip-flop movement of phospholipid along the lateral gate. Electrostatic contribution of the anionic phospholipids at the lateral gate may directly stabilize positively charged residues of the signal sequence of an incoming preprotein. Such a mechanism allows for the correct positioning of the entrant peptide, thereby providing a long-sought explanation for the role of anionic lipids in signal sequence folding during protein translocation.

Highlights

  • About 25–30% of bacterial proteins carry out their metabolic and structural function outside the cytoplasm

  • SecA initially associates with the membrane via a weak interaction of the SecA N-terminal amphipathic helix with PG, which induces a conformational change in SecA that enables subsequent high-affinity binding to the SecYEG channel

  • To verify the charge-dependency of this interaction, we investigated the influence of the anionic lipid species phosphatidylserine (PS), phosphatidic acid (PA) and cardiolipin (CL), and compared it with PG

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Summary

Introduction

About 25–30% of bacterial proteins carry out their metabolic and structural function outside the cytoplasm. They either have to be inserted into, or translocated across the cytoplasmic membrane. The major route for membrane protein insertion and translocation in bacteria is provided by the secretory (Sec) pathway. Targeting of proteins to the Sec translocase occurs either post-translationally via an amino-terminal (N-terminal) signal sequence or co-translationally as ribosome nascent chains with the aid of signal recognition particles (SRP) [1]. The SecB-preprotein complex is targeted to the motor ATPase SecA [2], which is bound to the membrane-embedded protein-conducting channel SecYEG and translocation is initiated

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