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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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The effect of FtsW in facilitating the transbilayer movement of Lipid II in model membranes is concentration dependent. Proteoliposomes were reconstituted in the presence of FtsW at the following protein/phospholipid molar ratio: 1:40 000 (1), 1:20 000 (2), 1:10 000 (3) and 1:5000 (4). The assay was performed as delineated under Figure 5. The percentage of quenching of fluorescence is dependent on the concentration of FtsW used in the reconstitution procedure. All measurements were carried out at 20°C and are representative of at least three independent experiments. A.U.: arbitrary units.
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f6: The effect of FtsW in facilitating the transbilayer movement of Lipid II in model membranes is concentration dependent. Proteoliposomes were reconstituted in the presence of FtsW at the following protein/phospholipid molar ratio: 1:40 000 (1), 1:20 000 (2), 1:10 000 (3) and 1:5000 (4). The assay was performed as delineated under Figure 5. The percentage of quenching of fluorescence is dependent on the concentration of FtsW used in the reconstitution procedure. All measurements were carried out at 20°C and are representative of at least three independent experiments. A.U.: arbitrary units.

Mentions: A completely different picture emerged when FtsW-containing proteoliposomes were assayed. With a protein to phospholipids molar ratio of ∼1:20 000, a reduction of ∼70% in fluorescence of NBD-Lipid II was visible, reflecting that only 30% of the NBD-labelled Lipid II remained protected from quenching by dithionite, suggesting that FtsW facilitated translocation of NBD-labelled Lipid II from the inner to the outer leaflet (Figure 5B, black trace). An intriguing observation is the persistence of a pool of protected NBD-labelled Lipid II, which can be explained as follows. The reconstitution procedure allows for the generation of a pool of NBD-Lipid II containing vesicles devoid of FtsW. Of these vesicles, only 50% of NBD-Lipid II is accessible to dithionite reduction. Therefore, the level of reduction by dithionite is expected to be dependent on the amount of transporters in the vesicle preparations, and the addition of more FtsW should result in fewer transporter-less LUVs. Indeed, the extent of quenching of fluorescence was shown to be FtsW concentration dependent, as measured from vesicles reconstituted with different amounts of FtsW (Figure 6). Yet, even at the highest amount of FtsW, a pool of NBD-labelled Lipid II remained protected from quenching by dithionite. This is best explained by aggregation of FtsW during the reconstitution procedure that is thereby not incorporated into the proteoliposomes and which would still allow for a significant amount of vesicles devoid of FtsW. These findings are consistent with results reported previously for translocation by other transporters of phospholipids and dolichol-linked oligosaccharides across the yeast endoplasmic reticulum (Vehring et al, 2007; Sanyal et al, 2008).


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)

The effect of FtsW in facilitating the transbilayer movement of Lipid II in model membranes is concentration dependent. Proteoliposomes were reconstituted in the presence of FtsW at the following protein/phospholipid molar ratio: 1:40 000 (1), 1:20 000 (2), 1:10 000 (3) and 1:5000 (4). The assay was performed as delineated under Figure 5. The percentage of quenching of fluorescence is dependent on the concentration of FtsW used in the reconstitution procedure. All measurements were carried out at 20°C and 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

f6: The effect of FtsW in facilitating the transbilayer movement of Lipid II in model membranes is concentration dependent. Proteoliposomes were reconstituted in the presence of FtsW at the following protein/phospholipid molar ratio: 1:40 000 (1), 1:20 000 (2), 1:10 000 (3) and 1:5000 (4). The assay was performed as delineated under Figure 5. The percentage of quenching of fluorescence is dependent on the concentration of FtsW used in the reconstitution procedure. All measurements were carried out at 20°C and are representative of at least three independent experiments. A.U.: arbitrary units.
Mentions: A completely different picture emerged when FtsW-containing proteoliposomes were assayed. With a protein to phospholipids molar ratio of ∼1:20 000, a reduction of ∼70% in fluorescence of NBD-Lipid II was visible, reflecting that only 30% of the NBD-labelled Lipid II remained protected from quenching by dithionite, suggesting that FtsW facilitated translocation of NBD-labelled Lipid II from the inner to the outer leaflet (Figure 5B, black trace). An intriguing observation is the persistence of a pool of protected NBD-labelled Lipid II, which can be explained as follows. The reconstitution procedure allows for the generation of a pool of NBD-Lipid II containing vesicles devoid of FtsW. Of these vesicles, only 50% of NBD-Lipid II is accessible to dithionite reduction. Therefore, the level of reduction by dithionite is expected to be dependent on the amount of transporters in the vesicle preparations, and the addition of more FtsW should result in fewer transporter-less LUVs. Indeed, the extent of quenching of fluorescence was shown to be FtsW concentration dependent, as measured from vesicles reconstituted with different amounts of FtsW (Figure 6). Yet, even at the highest amount of FtsW, a pool of NBD-labelled Lipid II remained protected from quenching by dithionite. This is best explained by aggregation of FtsW during the reconstitution procedure that is thereby not incorporated into the proteoliposomes and which would still allow for a significant amount of vesicles devoid of FtsW. These findings are consistent with results reported previously for translocation by other transporters of phospholipids and dolichol-linked oligosaccharides across the yeast endoplasmic reticulum (Vehring et al, 2007; Sanyal et al, 2008).

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