Specificity of the Transport of Lipid II by FtsW in Escherichia coli

Tamimount Mohammadi,Robert Sijbrandi,Mandy Lutters,Jolanda Verheul,Nathaniel I Martin,Tanneke Den Blaauwen,Ben De Kruijff,Eefjan Breukink

doi:10.1074/jbc.m114.557371

Abstract

Synthesis of biogenic membranes requires transbilayer movement of lipid-linked sugar molecules. This biological process, which is fundamental in prokaryotic cells, remains as yet not clearly understood. In order to obtain insights into the molecular basis of its mode of action, we analyzed the structure-function relationship between Lipid II, the important building block of the bacterial cell wall, and its inner membrane-localized transporter FtsW. Here, we show that the predicted transmembrane helix 4 of Escherichia coli FtsW (this protein consists of 10 predicted transmembrane segments) is required for the transport activity of the protein. We have identified two charged residues (Arg(145) and Lys(153)) within this segment that are specifically involved in the flipping of Lipid II. Mutating these two amino acids to uncharged ones affected the transport activity of FtsW. This was consistent with loss of in vivo activity of the mutants, as manifested by their inability to complement a temperature-sensitive strain of FtsW. The transport activity of FtsW could be inhibited with a Lipid II variant having an additional size of 420 Da. Reducing the size of this analog by about 274 Da resulted in the resumption of the transport activity of FtsW. This suggests that the integral membrane protein FtsW forms a size-restricted porelike structure, which accommodates Lipid II during transport across the bacterial cytoplasmic membrane.

Highlights

The mechanism of FtsW-mediated Lipid II transport across the bacterial cytoplasmic membrane is unknown
TM4 Is Required for the Transport Activity of FtsW—To determine which segments of the transporter are important for the flipping of Lipid II across the bacterial membrane, mutants lacking TM segments of E. coli FtsW were generated and are described above; they were shown to be important for interaction with proteins involved in cell division or cell wall synthesis
Using our reconstituted system, we were able to explore important questions in the transport process of the cell wall precursor Lipid II across the bacterial cytoplasmic membrane; we aimed to (i) identify residues that are required for the transport activity of FtsW and (ii) obtain insights into the nature of the “flipping device” that allows transbilayer movement of Lipid II

Summary

Introduction

The mechanism of FtsW-mediated Lipid II transport across the bacterial cytoplasmic membrane is unknown. Synthesis of biogenic membranes requires transbilayer movement of lipid-linked sugar molecules This biological process, which is fundamental in prokaryotic cells, remains as yet not clearly understood. We have identified two charged residues (Arg145 and Lys153) within this segment that are involved in the flipping of Lipid II. Mutating these two amino acids to uncharged ones affected the transport activity of FtsW. Reducing the size of this analog by about 274 Da resulted in the resumption of the transport activity of FtsW This suggests that the integral membrane protein FtsW forms a size-restricted porelike structure, which accommodates Lipid II during transport across the bacterial cytoplasmic membrane

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