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Identification of FtsW as a transporter of lipid-linked cell wall precursors across the membrane.

Mohammadi T, van Dam V, Sijbrandi R, Vernet T, Zapun A, Bouhss A, Diepeveen-de Bruin M, Nguyen-Distèche M, de Kruijff B, Breukink E - EMBO J. (2011)

Bottom Line: The intracellular part of the pathway results in the production of the membrane-anchored cell wall precursor, Lipid II.The translocation (flipping) step of Lipid II was demonstrated to require a specific protein (flippase).This study provides the first biochemical evidence for the involvement of an essential protein in the transport of lipid-linked cell wall precursors across biogenic membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Biology and Organic Chemistry, Institute of Biomembranes, Bijvoet Center for Biomolecular Research, Faculty of Science, Utrecht University, Padualaan, Utrecht, The Netherlands.

ABSTRACT
Bacterial cell growth necessitates synthesis of peptidoglycan. Assembly of this major constituent of the bacterial cell wall is a multistep process starting in the cytoplasm and ending in the exterior cell surface. The intracellular part of the pathway results in the production of the membrane-anchored cell wall precursor, Lipid II. After synthesis this lipid intermediate is translocated across the cell membrane. The translocation (flipping) step of Lipid II was demonstrated to require a specific protein (flippase). Here, we show that the integral membrane protein FtsW, an essential protein of the bacterial division machinery, is a transporter of the lipid-linked peptidoglycan precursors across the cytoplasmic membrane. Using Escherichia coli membrane vesicles we found that transport of Lipid II requires the presence of FtsW, and purified FtsW induced the transbilayer movement of Lipid II in model membranes. This study provides the first biochemical evidence for the involvement of an essential protein in the transport of lipid-linked cell wall precursors across biogenic membranes.

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Increased accessibility of Lipid II to vancomycin in proteoliposomes containing FtsW. Vesicles without any protein or reconstituted with the control protein KcsA or with FtsW were prepared according to the procedure described before. FRET measurements were then carried out at 14°C after addition of vancomycin-TMR. FtsW-containing vesicles display a much higher FRET signal than the protein-free and KcsA-containing vesicles. All measurements are representative of at least three independent experiments. A.U.: arbitrary units.
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f7: Increased accessibility of Lipid II to vancomycin in proteoliposomes containing FtsW. Vesicles without any protein or reconstituted with the control protein KcsA or with FtsW were prepared according to the procedure described before. FRET measurements were then carried out at 14°C after addition of vancomycin-TMR. FtsW-containing vesicles display a much higher FRET signal than the protein-free and KcsA-containing vesicles. All measurements are representative of at least three independent experiments. A.U.: arbitrary units.

Mentions: Our results using dithionite quenching were additionally confirmed using the FRET-based assay. We reconstituted vesicles in the absence of protein, with KcsA or FtsW. Addition of vancomycin-TMR yielded a higher FRET signal in the presence of FtsW than in the control situations; protein-free vesicles and vesicles reconstituted with KcsA (Figure 7). This result can be understood in the following way. Whereas in vesicles devoid of transporter (no protein or with KcsA), the vancomycin-TMR can bind to only half the pool of NBD-Lipid II, in vesicles harbouring FtsW, the vancomycin-TMR eventually binds to the most of the Lipid II pool that is flipped to the outer leaflet. Moreover, after subjecting these proteoliposomes to quenching by dithionite on ice, at a temperature that allows the reduction reaction but blocks the translocation, prior to the addition of vancomycin-TMR similar results were obtained (Supplementary Figure S7). Thus, a much larger Lipid II pool is accessible for vancomycin in the presence of FtsW, confirming its role as a Lipid II transporter.


Identification of FtsW as a transporter of lipid-linked cell wall precursors across the membrane.

Mohammadi T, van Dam V, Sijbrandi R, Vernet T, Zapun A, Bouhss A, Diepeveen-de Bruin M, Nguyen-Distèche M, de Kruijff B, Breukink E - EMBO J. (2011)

Increased accessibility of Lipid II to vancomycin in proteoliposomes containing FtsW. Vesicles without any protein or reconstituted with the control protein KcsA or with FtsW were prepared according to the procedure described before. FRET measurements were then carried out at 14°C after addition of vancomycin-TMR. FtsW-containing vesicles display a much higher FRET signal than the protein-free and KcsA-containing vesicles. All measurements are representative of at least three independent experiments. A.U.: arbitrary units.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3102273&req=5

f7: Increased accessibility of Lipid II to vancomycin in proteoliposomes containing FtsW. Vesicles without any protein or reconstituted with the control protein KcsA or with FtsW were prepared according to the procedure described before. FRET measurements were then carried out at 14°C after addition of vancomycin-TMR. FtsW-containing vesicles display a much higher FRET signal than the protein-free and KcsA-containing vesicles. All measurements are representative of at least three independent experiments. A.U.: arbitrary units.
Mentions: Our results using dithionite quenching were additionally confirmed using the FRET-based assay. We reconstituted vesicles in the absence of protein, with KcsA or FtsW. Addition of vancomycin-TMR yielded a higher FRET signal in the presence of FtsW than in the control situations; protein-free vesicles and vesicles reconstituted with KcsA (Figure 7). This result can be understood in the following way. Whereas in vesicles devoid of transporter (no protein or with KcsA), the vancomycin-TMR can bind to only half the pool of NBD-Lipid II, in vesicles harbouring FtsW, the vancomycin-TMR eventually binds to the most of the Lipid II pool that is flipped to the outer leaflet. Moreover, after subjecting these proteoliposomes to quenching by dithionite on ice, at a temperature that allows the reduction reaction but blocks the translocation, prior to the addition of vancomycin-TMR similar results were obtained (Supplementary Figure S7). Thus, a much larger Lipid II pool is accessible for vancomycin in the presence of FtsW, confirming its role as a Lipid II transporter.

Bottom Line: The intracellular part of the pathway results in the production of the membrane-anchored cell wall precursor, Lipid II.The translocation (flipping) step of Lipid II was demonstrated to require a specific protein (flippase).This study provides the first biochemical evidence for the involvement of an essential protein in the transport of lipid-linked cell wall precursors across biogenic membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Biology and Organic Chemistry, Institute of Biomembranes, Bijvoet Center for Biomolecular Research, Faculty of Science, Utrecht University, Padualaan, Utrecht, The Netherlands.

ABSTRACT
Bacterial cell growth necessitates synthesis of peptidoglycan. Assembly of this major constituent of the bacterial cell wall is a multistep process starting in the cytoplasm and ending in the exterior cell surface. The intracellular part of the pathway results in the production of the membrane-anchored cell wall precursor, Lipid II. After synthesis this lipid intermediate is translocated across the cell membrane. The translocation (flipping) step of Lipid II was demonstrated to require a specific protein (flippase). Here, we show that the integral membrane protein FtsW, an essential protein of the bacterial division machinery, is a transporter of the lipid-linked peptidoglycan precursors across the cytoplasmic membrane. Using Escherichia coli membrane vesicles we found that transport of Lipid II requires the presence of FtsW, and purified FtsW induced the transbilayer movement of Lipid II in model membranes. This study provides the first biochemical evidence for the involvement of an essential protein in the transport of lipid-linked cell wall precursors across biogenic membranes.

Show MeSH
Related in: MedlinePlus