Specificity of the transport of lipid II by FtsW in Escherichia coli.
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.
From Membrane Biochemistry and Biophysics, Department of Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Tamimount
T
Mohammadi
From Membrane Biochemistry and Biophysics, Department of Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands, Avans Hogeschool, Academie voor de Technologie van Gezondheid en Milieu, Lovensdijkstraat 61-63, 4818 AJ Breda, The Netherlands.
Robert
R
Sijbrandi
From Membrane Biochemistry and Biophysics, Department of Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Mandy
M
Lutters
Avans Hogeschool, Academie voor de Technologie van Gezondheid en Milieu, Lovensdijkstraat 61-63, 4818 AJ Breda, The Netherlands.
Jolanda
J
Verheul
the Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands, and.
Nathaniel I
NI
Martin
Bacterial Cell Biology, Swammerdam Institute of Life Sciences, Faculty of Science, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands.
Tanneke
T
den Blaauwen
From Membrane Biochemistry and Biophysics, Department of Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Ben
B
de Kruijff
From Membrane Biochemistry and Biophysics, Department of Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands, e.j.breukink@uu.nl.
Eefjan
E
Breukink
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D002473:Cell Wall / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D017433:Protein Structure, Secondary; D011510:Proteolipids / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D008565:Membrane Proteins; D011510:Proteolipids; D014538:Uridine Diphosphate N-Acetylmuramic Acid; C069249:muramyl-NAc-(pentapeptide)pyrophosphoryl-undecaprenol; C020485:proteoliposomes; C061684:FtsW protein, Bacteria; D049452:Green Fluorescent Proteins; D001120:Arginine; D008239:Lysine
Antibiotics; Cell Wall; Lipid Transport; Membrane Biogenesis; Phospholipid