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Localisation of DivIVA by targeting to negatively curved membranes.

Lenarcic R, Halbedel S, Visser L, Shaw M, Wu LJ, Errington J, Marenduzzo D, Hamoen LW - EMBO J. (2009)

Bottom Line: In mutants with aberrant cell shapes, DivIVA accumulates where the cell membrane is most strongly curved.On the basis of electron microscopic studies and other data, we propose that this is due to molecular bridging of the curvature by DivIVA multimers.A Monte-Carlo simulation study showed that molecular bridging can be a general mechanism for binding of proteins to negatively curved membranes.

View Article: PubMed Central - PubMed

Affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne, UK.

ABSTRACT
DivIVA is a conserved protein in Gram-positive bacteria and involved in various processes related to cell growth, cell division and spore formation. DivIVA is specifically targeted to cell division sites and cell poles. In Bacillus subtilis, DivIVA helps to localise other proteins, such as the conserved cell division inhibitor proteins, MinC/MinD, and the chromosome segregation protein, RacA. Little is known about the mechanism that localises DivIVA. Here we show that DivIVA binds to liposomes, and that the N terminus harbours the membrane targeting sequence. The purified protein can stimulate binding of RacA to membranes. In mutants with aberrant cell shapes, DivIVA accumulates where the cell membrane is most strongly curved. On the basis of electron microscopic studies and other data, we propose that this is due to molecular bridging of the curvature by DivIVA multimers. This model may explain why DivIVA localises at cell division sites. A Monte-Carlo simulation study showed that molecular bridging can be a general mechanism for binding of proteins to negatively curved membranes.

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Binding of purified GFP (A), DivIVA-GFP (B) and DivIVA (C) to liposomes adhered to a Biacore L1 sensor chip. Protein samples were injected (a), and after ∼2 min, followed by an injection of buffer alone (b). The chip was regenerated by a short injection (c and d) with 0.1 M NaOH solution. The flow rate was 30 μl/min, and protein concentrations were 3.1, 1.1 and 1.5 mg/ml for GFP, DivIVA-GFP and DivIVA, respectively. The response is given in artificial resonance units (RU). (D–G) SPR analysis of DivIVA deletions that were purified as MBP fusions. The C-terminal deletion (ΔC-DivIVA-MBP) lacks the last 20 amino acids of DivIVA and the N-terminal deletion (ΔN-DivIVA-MBP) lacks the first 40 amino acids of DivIVA. Protein concentrations were 0.4 mg/ml. (H) Sedimentation analyses of the N- and C-terminal DivIVA deletions in the presence and absence of liposomes. The total fraction, before centrifugation (T), and the supernatant (S) and pellet (P) fractions after centrifugation, was analysed by SDS–PAGE.
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f3: Binding of purified GFP (A), DivIVA-GFP (B) and DivIVA (C) to liposomes adhered to a Biacore L1 sensor chip. Protein samples were injected (a), and after ∼2 min, followed by an injection of buffer alone (b). The chip was regenerated by a short injection (c and d) with 0.1 M NaOH solution. The flow rate was 30 μl/min, and protein concentrations were 3.1, 1.1 and 1.5 mg/ml for GFP, DivIVA-GFP and DivIVA, respectively. The response is given in artificial resonance units (RU). (D–G) SPR analysis of DivIVA deletions that were purified as MBP fusions. The C-terminal deletion (ΔC-DivIVA-MBP) lacks the last 20 amino acids of DivIVA and the N-terminal deletion (ΔN-DivIVA-MBP) lacks the first 40 amino acids of DivIVA. Protein concentrations were 0.4 mg/ml. (H) Sedimentation analyses of the N- and C-terminal DivIVA deletions in the presence and absence of liposomes. The total fraction, before centrifugation (T), and the supernatant (S) and pellet (P) fractions after centrifugation, was analysed by SDS–PAGE.

Mentions: As an independent way to test the affinity of DivIVA for lipids, we turned to surface plasmon resonance (SPR). Liposomes were adsorbed onto an L1 sensor chip and purified GFP, DivIVA-GFP, or DivIVA was injected. As shown in the sensograms of Figure 3A, the addition of GFP yielded only a small response (a), likely due to a buffer effect given that the signal dropped back to the baseline when the GFP injection ended (b). In contrast, the magnitude of the response was strong when DivIVA-GFP was injected (Figure 3B), and the signal remained when the flow of DivIVA-GFP ceased, indicating that the fusion protein strongly interacted with the phospholipid membranes (the initial sharp decrease was due to buffer effects). Induction of DivIVA (Figure 3C) also resulted in a strong response and, together with the low off-rate, confirmed that this protein binds specifically to liposomes. Owing to the complex oligomerisation characteristics of DivIVA, it is difficult to deduce kinetic parameters from these sensograms. However, the difference in off-rates between DivIVA and DivIVA-GFP suggests that the GFP-tag influences membrane binding.


Localisation of DivIVA by targeting to negatively curved membranes.

Lenarcic R, Halbedel S, Visser L, Shaw M, Wu LJ, Errington J, Marenduzzo D, Hamoen LW - EMBO J. (2009)

Binding of purified GFP (A), DivIVA-GFP (B) and DivIVA (C) to liposomes adhered to a Biacore L1 sensor chip. Protein samples were injected (a), and after ∼2 min, followed by an injection of buffer alone (b). The chip was regenerated by a short injection (c and d) with 0.1 M NaOH solution. The flow rate was 30 μl/min, and protein concentrations were 3.1, 1.1 and 1.5 mg/ml for GFP, DivIVA-GFP and DivIVA, respectively. The response is given in artificial resonance units (RU). (D–G) SPR analysis of DivIVA deletions that were purified as MBP fusions. The C-terminal deletion (ΔC-DivIVA-MBP) lacks the last 20 amino acids of DivIVA and the N-terminal deletion (ΔN-DivIVA-MBP) lacks the first 40 amino acids of DivIVA. Protein concentrations were 0.4 mg/ml. (H) Sedimentation analyses of the N- and C-terminal DivIVA deletions in the presence and absence of liposomes. The total fraction, before centrifugation (T), and the supernatant (S) and pellet (P) fractions after centrifugation, was analysed by SDS–PAGE.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2690451&req=5

f3: Binding of purified GFP (A), DivIVA-GFP (B) and DivIVA (C) to liposomes adhered to a Biacore L1 sensor chip. Protein samples were injected (a), and after ∼2 min, followed by an injection of buffer alone (b). The chip was regenerated by a short injection (c and d) with 0.1 M NaOH solution. The flow rate was 30 μl/min, and protein concentrations were 3.1, 1.1 and 1.5 mg/ml for GFP, DivIVA-GFP and DivIVA, respectively. The response is given in artificial resonance units (RU). (D–G) SPR analysis of DivIVA deletions that were purified as MBP fusions. The C-terminal deletion (ΔC-DivIVA-MBP) lacks the last 20 amino acids of DivIVA and the N-terminal deletion (ΔN-DivIVA-MBP) lacks the first 40 amino acids of DivIVA. Protein concentrations were 0.4 mg/ml. (H) Sedimentation analyses of the N- and C-terminal DivIVA deletions in the presence and absence of liposomes. The total fraction, before centrifugation (T), and the supernatant (S) and pellet (P) fractions after centrifugation, was analysed by SDS–PAGE.
Mentions: As an independent way to test the affinity of DivIVA for lipids, we turned to surface plasmon resonance (SPR). Liposomes were adsorbed onto an L1 sensor chip and purified GFP, DivIVA-GFP, or DivIVA was injected. As shown in the sensograms of Figure 3A, the addition of GFP yielded only a small response (a), likely due to a buffer effect given that the signal dropped back to the baseline when the GFP injection ended (b). In contrast, the magnitude of the response was strong when DivIVA-GFP was injected (Figure 3B), and the signal remained when the flow of DivIVA-GFP ceased, indicating that the fusion protein strongly interacted with the phospholipid membranes (the initial sharp decrease was due to buffer effects). Induction of DivIVA (Figure 3C) also resulted in a strong response and, together with the low off-rate, confirmed that this protein binds specifically to liposomes. Owing to the complex oligomerisation characteristics of DivIVA, it is difficult to deduce kinetic parameters from these sensograms. However, the difference in off-rates between DivIVA and DivIVA-GFP suggests that the GFP-tag influences membrane binding.

Bottom Line: In mutants with aberrant cell shapes, DivIVA accumulates where the cell membrane is most strongly curved.On the basis of electron microscopic studies and other data, we propose that this is due to molecular bridging of the curvature by DivIVA multimers.A Monte-Carlo simulation study showed that molecular bridging can be a general mechanism for binding of proteins to negatively curved membranes.

View Article: PubMed Central - PubMed

Affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne, UK.

ABSTRACT
DivIVA is a conserved protein in Gram-positive bacteria and involved in various processes related to cell growth, cell division and spore formation. DivIVA is specifically targeted to cell division sites and cell poles. In Bacillus subtilis, DivIVA helps to localise other proteins, such as the conserved cell division inhibitor proteins, MinC/MinD, and the chromosome segregation protein, RacA. Little is known about the mechanism that localises DivIVA. Here we show that DivIVA binds to liposomes, and that the N terminus harbours the membrane targeting sequence. The purified protein can stimulate binding of RacA to membranes. In mutants with aberrant cell shapes, DivIVA accumulates where the cell membrane is most strongly curved. On the basis of electron microscopic studies and other data, we propose that this is due to molecular bridging of the curvature by DivIVA multimers. This model may explain why DivIVA localises at cell division sites. A Monte-Carlo simulation study showed that molecular bridging can be a general mechanism for binding of proteins to negatively curved membranes.

Show MeSH
Related in: MedlinePlus