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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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Sedimentation reactions of purified FtsZ and FtsZ CTV mutant proteins with ZapD.A. FtsZ and FtsZ CTV mutants (5 ∝M) were incubated alone or combined with purified ZapD at 1:0.2 or 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) containing 3 ∝M BSA. Reactions were processed as outlined in the Materials and Methods section in the main text. Equivalent aliquots (5 ∝l) of pellet (bottom panel) and supernatants (top panel) were resolved on a 12.5% SDS-PAGE gel and stained with SimplyBlue SafeStain (Invitrogen). A representative gel image of three independent experiments is shown. B. The amounts of FtsZ or FtsZ CTV mutant proteins present in the pellet fractions in reactions with or without ZapD are reported as a percentage. The average numbers and standard deviation bars are from at least three independent experiments. Of note, FtsZ CTV containing NRNKRG sequences show the highest pelletable amounts of FtsZ under the experimental conditions of this study. C. The amounts of ZapD protein present in the pellet fractions in reactions with FtsZ or FtsZ CTV mutants are reported as a percentage. The average numbers and standard deviation bars are from at least three independent experiments.
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pone.0153337.g002: Sedimentation reactions of purified FtsZ and FtsZ CTV mutant proteins with ZapD.A. FtsZ and FtsZ CTV mutants (5 ∝M) were incubated alone or combined with purified ZapD at 1:0.2 or 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) containing 3 ∝M BSA. Reactions were processed as outlined in the Materials and Methods section in the main text. Equivalent aliquots (5 ∝l) of pellet (bottom panel) and supernatants (top panel) were resolved on a 12.5% SDS-PAGE gel and stained with SimplyBlue SafeStain (Invitrogen). A representative gel image of three independent experiments is shown. B. The amounts of FtsZ or FtsZ CTV mutant proteins present in the pellet fractions in reactions with or without ZapD are reported as a percentage. The average numbers and standard deviation bars are from at least three independent experiments. Of note, FtsZ CTV containing NRNKRG sequences show the highest pelletable amounts of FtsZ under the experimental conditions of this study. C. The amounts of ZapD protein present in the pellet fractions in reactions with FtsZ or FtsZ CTV mutants are reported as a percentage. The average numbers and standard deviation bars are from at least three independent experiments.

Mentions: When incubated with GTP, 26 ± 8% of WT FtsZ and similar amounts of FtsZ1-379 and other FtsZ CTV variants, except the NRNKRG variant were present in the pellet (Fig 2). It has been previously reported that an E. coli FtsZ chimeric construct containing a B. subtilis CTV sequence (NRNKRG) shows enhanced bundling compared to E. coli FtsZ [22]. In the presence of ZapD, ~2–3 fold increases in amounts of WT FtsZ were noted together with ~55–62% of ZapD recovered in the pellet. ZapD alone was barely detectable after sedimentation in the presence of GTP (Fig 2). These data are consistent with an increase in the sedimentable WT FtsZ polymer mass. However, no significant changes in pelletable amounts of FtsZ without the CTV region (FtsZ1-379) were observed with or without ZapD, nor did significant amounts of ZapD co-sediment with FtsZ1-379 (Fig 2).


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)

Sedimentation reactions of purified FtsZ and FtsZ CTV mutant proteins with ZapD.A. FtsZ and FtsZ CTV mutants (5 ∝M) were incubated alone or combined with purified ZapD at 1:0.2 or 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) containing 3 ∝M BSA. Reactions were processed as outlined in the Materials and Methods section in the main text. Equivalent aliquots (5 ∝l) of pellet (bottom panel) and supernatants (top panel) were resolved on a 12.5% SDS-PAGE gel and stained with SimplyBlue SafeStain (Invitrogen). A representative gel image of three independent experiments is shown. B. The amounts of FtsZ or FtsZ CTV mutant proteins present in the pellet fractions in reactions with or without ZapD are reported as a percentage. The average numbers and standard deviation bars are from at least three independent experiments. Of note, FtsZ CTV containing NRNKRG sequences show the highest pelletable amounts of FtsZ under the experimental conditions of this study. C. The amounts of ZapD protein present in the pellet fractions in reactions with FtsZ or FtsZ CTV mutants are reported as a percentage. The average numbers and standard deviation bars are from at least three independent experiments.
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pone.0153337.g002: Sedimentation reactions of purified FtsZ and FtsZ CTV mutant proteins with ZapD.A. FtsZ and FtsZ CTV mutants (5 ∝M) were incubated alone or combined with purified ZapD at 1:0.2 or 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) containing 3 ∝M BSA. Reactions were processed as outlined in the Materials and Methods section in the main text. Equivalent aliquots (5 ∝l) of pellet (bottom panel) and supernatants (top panel) were resolved on a 12.5% SDS-PAGE gel and stained with SimplyBlue SafeStain (Invitrogen). A representative gel image of three independent experiments is shown. B. The amounts of FtsZ or FtsZ CTV mutant proteins present in the pellet fractions in reactions with or without ZapD are reported as a percentage. The average numbers and standard deviation bars are from at least three independent experiments. Of note, FtsZ CTV containing NRNKRG sequences show the highest pelletable amounts of FtsZ under the experimental conditions of this study. C. The amounts of ZapD protein present in the pellet fractions in reactions with FtsZ or FtsZ CTV mutants are reported as a percentage. The average numbers and standard deviation bars are from at least three independent experiments.
Mentions: When incubated with GTP, 26 ± 8% of WT FtsZ and similar amounts of FtsZ1-379 and other FtsZ CTV variants, except the NRNKRG variant were present in the pellet (Fig 2). It has been previously reported that an E. coli FtsZ chimeric construct containing a B. subtilis CTV sequence (NRNKRG) shows enhanced bundling compared to E. coli FtsZ [22]. In the presence of ZapD, ~2–3 fold increases in amounts of WT FtsZ were noted together with ~55–62% of ZapD recovered in the pellet. ZapD alone was barely detectable after sedimentation in the presence of GTP (Fig 2). These data are consistent with an increase in the sedimentable WT FtsZ polymer mass. However, no significant changes in pelletable amounts of FtsZ without the CTV region (FtsZ1-379) were observed with or without ZapD, nor did significant amounts of ZapD co-sediment with FtsZ1-379 (Fig 2).

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