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Characterization of the FtsZ C-Terminal Variable (CTV) Region in Z-Ring Assembly and Interaction with the Z-Ring Stabilizer ZapD in E. coli Cytokinesis.

Huang KH, Mychack A, Tchorzewski L, Janakiraman A - PLoS ONE (2016)

Bottom Line: Multiple Z-ring associated proteins (Zaps), also promote lateral interactions between FtsZ protofilaments to stabilize the FtsZ ring in vivo.Our data suggest a mechanism in which the CTV residues, particularly K380, facilitate a conformation for the conserved carboxy-terminal residues in FtsZ, that lie immediately N-terminal to the CTV, to enable optimal contact with ZapD.Further, phylogenetic analyses suggest a correlation between the nature of FtsZ CTV residues and the presence of ZapD in the β- γ-proteobacterial species.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, City College of CUNY, 160 Convent Avenue, MR 526, New York, NY, United States of America.

ABSTRACT
Polymerization of a ring-like cytoskeletal structure, the Z-ring, at midcell is a highly conserved feature in virtually all bacteria. The Z-ring is composed of short protofilaments of the tubulin homolog FtsZ, randomly arranged and held together through lateral interactions. In vitro, lateral associations between FtsZ protofilaments are stabilized by crowding agents, high concentrations of divalent cations, or in some cases, low pH. In vivo, the last 4-10 amino acid residues at the C-terminus of FtsZ (the C-terminal variable region, CTV) have been implicated in mediating lateral associations between FtsZ protofilaments through charge shielding. Multiple Z-ring associated proteins (Zaps), also promote lateral interactions between FtsZ protofilaments to stabilize the FtsZ ring in vivo. Here we characterize the complementary role/s of the CTV of E. coli FtsZ and the FtsZ-ring stabilizing protein ZapD, in FtsZ assembly. We show that the net charge of the FtsZ CTV not only affects FtsZ protofilament bundling, confirming earlier observations, but likely also the length of the FtsZ protofilaments in vitro. The CTV residues also have important consequences for Z-ring assembly and interaction with ZapD in the cell. ZapD requires the FtsZ CTV region for interaction with FtsZ in vitro and for localization to midcell in vivo. Our data suggest a mechanism in which the CTV residues, particularly K380, facilitate a conformation for the conserved carboxy-terminal residues in FtsZ, that lie immediately N-terminal to the CTV, to enable optimal contact with ZapD. Further, phylogenetic analyses suggest a correlation between the nature of FtsZ CTV residues and the presence of ZapD in the β- γ-proteobacterial species.

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Morphologies of polymeric assemblies of FtsZ and FtsZ CTV mutant proteins.In vitro reactions containing FtsZ and FtsZ CTV mutants (5 ∝M) alone or combined with purified ZapD at 1:1 ratios in a polymerization buffer (50 mM K-MOPS pH 6.5, 50 mM KCl, 2.5 mM MgCl2, and 1 mM GTP) were incubated for 5 mins at room temperature. A 10-μl aliquot of each reaction was placed on carbon-coated copper grids (Electron Microscopy Sciences), processed and imaged as described in the material and methods section of the main text. Negative stained transmission electron microscopy images of FtsZ or FtsZ CTV mutants with or without ZapD are shown. Bar = 200 nm.
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pone.0153337.g003: Morphologies of polymeric assemblies of FtsZ and FtsZ CTV mutant proteins.In vitro reactions containing FtsZ and FtsZ CTV mutants (5 ∝M) alone or combined with purified ZapD at 1:1 ratios in a polymerization buffer (50 mM K-MOPS pH 6.5, 50 mM KCl, 2.5 mM MgCl2, and 1 mM GTP) were incubated for 5 mins at room temperature. A 10-μl aliquot of each reaction was placed on carbon-coated copper grids (Electron Microscopy Sciences), processed and imaged as described in the material and methods section of the main text. Negative stained transmission electron microscopy images of FtsZ or FtsZ CTV mutants with or without ZapD are shown. Bar = 200 nm.

Mentions: We observed that WT FtsZ shows typical single protofilaments in the presence of GTP and polymeric bundles upon addition of ZapD as previously reported in the literature (Fig 3) [25]. As expected, no FtsZ protofilaments were observed in the absence of GTP (data not shown). To visualize the FtsZ CTV mutants alone or in the presence of ZapD, we used equimolar ratios of ZapD and FtsZ, as the co-sedimentation profiles of ZapD at lower concentration (1 μM) were not significantly different than those at higher concentration (5 μM) (Fig 2). FtsZ missing the CTV sequences (FtsZ1-379) forms single protofilaments that are slightly shorter than WT FtsZ, confirming an earlier observation by Buske and Levin (Fig 3 and S3 Fig) [22]. Addition of ZapD fails to promote bundling of FtsZ1-379 polymers (Fig 3 and S3 Fig). The net-neutral DQAK mutant showed largely single protofilaments similar to WT FtsZ, while the QQQQ mutant displayed modest amounts of lateral bundling on its own, perhaps due to the tendency of poly-glutamine sequences to aggregate (Fig 3). This suggests that a net-neutral FtsZ CTV retains the ability to maintain a functional interaction with ZapD as in the presence of ZapD, both FtsZ variants showed significant increases in bundled forms similar to WT FtsZ (Fig 3). The net-negative DQAD mutant displayed single protofilaments in the presence of GTP and no detectable changes in polymerization were observed upon addition of ZapD (Fig 3).


Characterization of the FtsZ C-Terminal Variable (CTV) Region in Z-Ring Assembly and Interaction with the Z-Ring Stabilizer ZapD in E. coli Cytokinesis.

Huang KH, Mychack A, Tchorzewski L, Janakiraman A - PLoS ONE (2016)

Morphologies of polymeric assemblies of FtsZ and FtsZ CTV mutant proteins.In vitro reactions containing FtsZ and FtsZ CTV mutants (5 ∝M) alone or combined with purified ZapD at 1:1 ratios in a polymerization buffer (50 mM K-MOPS pH 6.5, 50 mM KCl, 2.5 mM MgCl2, and 1 mM GTP) were incubated for 5 mins at room temperature. A 10-μl aliquot of each reaction was placed on carbon-coated copper grids (Electron Microscopy Sciences), processed and imaged as described in the material and methods section of the main text. Negative stained transmission electron microscopy images of FtsZ or FtsZ CTV mutants with or without ZapD are shown. Bar = 200 nm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835091&req=5

pone.0153337.g003: Morphologies of polymeric assemblies of FtsZ and FtsZ CTV mutant proteins.In vitro reactions containing FtsZ and FtsZ CTV mutants (5 ∝M) alone or combined with purified ZapD at 1:1 ratios in a polymerization buffer (50 mM K-MOPS pH 6.5, 50 mM KCl, 2.5 mM MgCl2, and 1 mM GTP) were incubated for 5 mins at room temperature. A 10-μl aliquot of each reaction was placed on carbon-coated copper grids (Electron Microscopy Sciences), processed and imaged as described in the material and methods section of the main text. Negative stained transmission electron microscopy images of FtsZ or FtsZ CTV mutants with or without ZapD are shown. Bar = 200 nm.
Mentions: We observed that WT FtsZ shows typical single protofilaments in the presence of GTP and polymeric bundles upon addition of ZapD as previously reported in the literature (Fig 3) [25]. As expected, no FtsZ protofilaments were observed in the absence of GTP (data not shown). To visualize the FtsZ CTV mutants alone or in the presence of ZapD, we used equimolar ratios of ZapD and FtsZ, as the co-sedimentation profiles of ZapD at lower concentration (1 μM) were not significantly different than those at higher concentration (5 μM) (Fig 2). FtsZ missing the CTV sequences (FtsZ1-379) forms single protofilaments that are slightly shorter than WT FtsZ, confirming an earlier observation by Buske and Levin (Fig 3 and S3 Fig) [22]. Addition of ZapD fails to promote bundling of FtsZ1-379 polymers (Fig 3 and S3 Fig). The net-neutral DQAK mutant showed largely single protofilaments similar to WT FtsZ, while the QQQQ mutant displayed modest amounts of lateral bundling on its own, perhaps due to the tendency of poly-glutamine sequences to aggregate (Fig 3). This suggests that a net-neutral FtsZ CTV retains the ability to maintain a functional interaction with ZapD as in the presence of ZapD, both FtsZ variants showed significant increases in bundled forms similar to WT FtsZ (Fig 3). The net-negative DQAD mutant displayed single protofilaments in the presence of GTP and no detectable changes in polymerization were observed upon addition of ZapD (Fig 3).

Bottom Line: Multiple Z-ring associated proteins (Zaps), also promote lateral interactions between FtsZ protofilaments to stabilize the FtsZ ring in vivo.Our data suggest a mechanism in which the CTV residues, particularly K380, facilitate a conformation for the conserved carboxy-terminal residues in FtsZ, that lie immediately N-terminal to the CTV, to enable optimal contact with ZapD.Further, phylogenetic analyses suggest a correlation between the nature of FtsZ CTV residues and the presence of ZapD in the β- γ-proteobacterial species.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, City College of CUNY, 160 Convent Avenue, MR 526, New York, NY, United States of America.

ABSTRACT
Polymerization of a ring-like cytoskeletal structure, the Z-ring, at midcell is a highly conserved feature in virtually all bacteria. The Z-ring is composed of short protofilaments of the tubulin homolog FtsZ, randomly arranged and held together through lateral interactions. In vitro, lateral associations between FtsZ protofilaments are stabilized by crowding agents, high concentrations of divalent cations, or in some cases, low pH. In vivo, the last 4-10 amino acid residues at the C-terminus of FtsZ (the C-terminal variable region, CTV) have been implicated in mediating lateral associations between FtsZ protofilaments through charge shielding. Multiple Z-ring associated proteins (Zaps), also promote lateral interactions between FtsZ protofilaments to stabilize the FtsZ ring in vivo. Here we characterize the complementary role/s of the CTV of E. coli FtsZ and the FtsZ-ring stabilizing protein ZapD, in FtsZ assembly. We show that the net charge of the FtsZ CTV not only affects FtsZ protofilament bundling, confirming earlier observations, but likely also the length of the FtsZ protofilaments in vitro. The CTV residues also have important consequences for Z-ring assembly and interaction with ZapD in the cell. ZapD requires the FtsZ CTV region for interaction with FtsZ in vitro and for localization to midcell in vivo. Our data suggest a mechanism in which the CTV residues, particularly K380, facilitate a conformation for the conserved carboxy-terminal residues in FtsZ, that lie immediately N-terminal to the CTV, to enable optimal contact with ZapD. Further, phylogenetic analyses suggest a correlation between the nature of FtsZ CTV residues and the presence of ZapD in the β- γ-proteobacterial species.

Show MeSH
Related in: MedlinePlus