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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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Bacterial two-hybrid interaction assay (A). divIVA and racA were cloned in different expression vectors, and the combinations screened for adenylate cyclase activity (blue colonies). A positive interaction was observed with a DivIVA-adenylate cyclase T25 fragment on the low-copy plasmid p25-N and an adenylate cyclase T18 fragment-RacA fusion on the high-copy plasmid pUT18C. pKT25: low-copy plasmid for N-terminal adenylate cyclase T25 fragment fusion, p25-N: low-copy plasmid for C-terminal adenylate cyclase fusion, pUT18C: high-copy plasmid for N-terminal adenylate cyclase T18 fragment fusion, pUT18: high-copy plasmid for C-terminal adenylate cyclase T18 fragment fusion. The effect of DivIVA on the binding of MBP-RacA to liposomes (B). MBP-RacA (6.4 ng) and DivIVA (3.6 μg) were mixed with liposomes (90 μg) in a sucrose-containing buffer, and loaded at the bottom of a sucrose gradient. After centrifugation, gradients where sampled in five fractions (top fractions (low density) to bottom fractions (high density) run from left to right). Liposomes floated to the two top fractions and where clearly visible. Gradient fractions were analysed by western blotting using RacA-specific antibodies.
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f7: Bacterial two-hybrid interaction assay (A). divIVA and racA were cloned in different expression vectors, and the combinations screened for adenylate cyclase activity (blue colonies). A positive interaction was observed with a DivIVA-adenylate cyclase T25 fragment on the low-copy plasmid p25-N and an adenylate cyclase T18 fragment-RacA fusion on the high-copy plasmid pUT18C. pKT25: low-copy plasmid for N-terminal adenylate cyclase T25 fragment fusion, p25-N: low-copy plasmid for C-terminal adenylate cyclase fusion, pUT18C: high-copy plasmid for N-terminal adenylate cyclase T18 fragment fusion, pUT18: high-copy plasmid for C-terminal adenylate cyclase T18 fragment fusion. The effect of DivIVA on the binding of MBP-RacA to liposomes (B). MBP-RacA (6.4 ng) and DivIVA (3.6 μg) were mixed with liposomes (90 μg) in a sucrose-containing buffer, and loaded at the bottom of a sucrose gradient. After centrifugation, gradients where sampled in five fractions (top fractions (low density) to bottom fractions (high density) run from left to right). Liposomes floated to the two top fractions and where clearly visible. Gradient fractions were analysed by western blotting using RacA-specific antibodies.

Mentions: DivIVA is required for the localisation of proteins, but so far there is no biochemical data showing a direct interaction between DivIVA and other proteins. We were unable to detect any effect on the lipid-binding affinity of purified MinD by DivIVA (data not shown). Very recently, it was reported that MinJ (YvjD), a previously unknown transmembrane protein, is required for MinD localisation (Bramkamp et al, 2008; Patrick and Kearns, 2008), which explains our failure to show a direct interaction between DivIVA and MinD in vitro. Another potential target for DivIVA interaction is the chromosome segregation protein, RacA (Ben-Yehuda et al, 2003). We first used a bacterial two-hybrid assay to obtain more evidence for a direct interaction between DivIVA and RacA. Such a test gave a negative outcome in case of MinD and DivIVA (data not shown). We tested different combinations of both C- and N-terminal fusions, and low- and high-copy vectors (Supplementary data), and found a positive interaction between a DivIVA-adenylate cyclase T25 fragment fusion on a low-copy plasmid and an adenylate cyclase T18 fragment-RacA fusion on a high-copy plasmid (Figure 7A). Encouraged by this result, we purified RacA as a fusion with MBP. We used density gradient flotation experiments to test whether DivIVA would stimulate binding of RacA to lipid membranes. MBP-RacA was mixed with DivIVA and liposomes, and loaded at the bottom of a sucrose gradient. A high concentration of BSA (0.5 mg/ml) was present to ensure specificity. After centrifugation, fractions were loaded onto a protein gel and analysed by western blotting using RacA-specific antibodies (Figure 7B). In the absence of DivIVA, a small amount of RacA could be detected in the phospholipid fractions. However, the presence of DivIVA led indeed to a substantial increase in the amount of RacA in the lipid fraction.


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)

Bacterial two-hybrid interaction assay (A). divIVA and racA were cloned in different expression vectors, and the combinations screened for adenylate cyclase activity (blue colonies). A positive interaction was observed with a DivIVA-adenylate cyclase T25 fragment on the low-copy plasmid p25-N and an adenylate cyclase T18 fragment-RacA fusion on the high-copy plasmid pUT18C. pKT25: low-copy plasmid for N-terminal adenylate cyclase T25 fragment fusion, p25-N: low-copy plasmid for C-terminal adenylate cyclase fusion, pUT18C: high-copy plasmid for N-terminal adenylate cyclase T18 fragment fusion, pUT18: high-copy plasmid for C-terminal adenylate cyclase T18 fragment fusion. The effect of DivIVA on the binding of MBP-RacA to liposomes (B). MBP-RacA (6.4 ng) and DivIVA (3.6 μg) were mixed with liposomes (90 μg) in a sucrose-containing buffer, and loaded at the bottom of a sucrose gradient. After centrifugation, gradients where sampled in five fractions (top fractions (low density) to bottom fractions (high density) run from left to right). Liposomes floated to the two top fractions and where clearly visible. Gradient fractions were analysed by western blotting using RacA-specific antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Bacterial two-hybrid interaction assay (A). divIVA and racA were cloned in different expression vectors, and the combinations screened for adenylate cyclase activity (blue colonies). A positive interaction was observed with a DivIVA-adenylate cyclase T25 fragment on the low-copy plasmid p25-N and an adenylate cyclase T18 fragment-RacA fusion on the high-copy plasmid pUT18C. pKT25: low-copy plasmid for N-terminal adenylate cyclase T25 fragment fusion, p25-N: low-copy plasmid for C-terminal adenylate cyclase fusion, pUT18C: high-copy plasmid for N-terminal adenylate cyclase T18 fragment fusion, pUT18: high-copy plasmid for C-terminal adenylate cyclase T18 fragment fusion. The effect of DivIVA on the binding of MBP-RacA to liposomes (B). MBP-RacA (6.4 ng) and DivIVA (3.6 μg) were mixed with liposomes (90 μg) in a sucrose-containing buffer, and loaded at the bottom of a sucrose gradient. After centrifugation, gradients where sampled in five fractions (top fractions (low density) to bottom fractions (high density) run from left to right). Liposomes floated to the two top fractions and where clearly visible. Gradient fractions were analysed by western blotting using RacA-specific antibodies.
Mentions: DivIVA is required for the localisation of proteins, but so far there is no biochemical data showing a direct interaction between DivIVA and other proteins. We were unable to detect any effect on the lipid-binding affinity of purified MinD by DivIVA (data not shown). Very recently, it was reported that MinJ (YvjD), a previously unknown transmembrane protein, is required for MinD localisation (Bramkamp et al, 2008; Patrick and Kearns, 2008), which explains our failure to show a direct interaction between DivIVA and MinD in vitro. Another potential target for DivIVA interaction is the chromosome segregation protein, RacA (Ben-Yehuda et al, 2003). We first used a bacterial two-hybrid assay to obtain more evidence for a direct interaction between DivIVA and RacA. Such a test gave a negative outcome in case of MinD and DivIVA (data not shown). We tested different combinations of both C- and N-terminal fusions, and low- and high-copy vectors (Supplementary data), and found a positive interaction between a DivIVA-adenylate cyclase T25 fragment fusion on a low-copy plasmid and an adenylate cyclase T18 fragment-RacA fusion on a high-copy plasmid (Figure 7A). Encouraged by this result, we purified RacA as a fusion with MBP. We used density gradient flotation experiments to test whether DivIVA would stimulate binding of RacA to lipid membranes. MBP-RacA was mixed with DivIVA and liposomes, and loaded at the bottom of a sucrose gradient. A high concentration of BSA (0.5 mg/ml) was present to ensure specificity. After centrifugation, fractions were loaded onto a protein gel and analysed by western blotting using RacA-specific antibodies (Figure 7B). In the absence of DivIVA, a small amount of RacA could be detected in the phospholipid fractions. However, the presence of DivIVA led indeed to a substantial increase in the amount of RacA in the lipid fraction.

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