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. 2001 Dec;183(24):7190-7.
doi: 10.1128/JB.183.24.7190-7197.2001.

Assembly of an FtsZ mutant deficient in GTPase activity has implications for FtsZ assembly and the role of the Z ring in cell division

A Mukherjee  1 , C Saez, J Lutkenhaus
Affiliations

Assembly of an FtsZ mutant deficient in GTPase activity has implications for FtsZ assembly and the role of the Z ring in cell division

A Mukherjee et al. J Bacteriol. 2001 Dec.

Abstract

FtsZ, the ancestral homologue of eukaryotic tubulins, assembles into the Z ring, which is required for cytokinesis in prokaryotic cells. Both FtsZ and tubulin have a GTPase activity associated with polymerization. Interestingly, the ftsZ2 mutant is viable, although the FtsZ2 mutant protein has dramatically reduced GTPase activity due to a glycine-for-aspartic acid substitution within the synergy loop. In this study, we have examined the properties of FtsZ2 and found that the reduced GTPase activity is not enhanced by DEAE-dextran-induced assembly, indicating it has a defective catalytic site. In the absence of DEAE-dextran, FtsZ2 fails to assemble unless supplemented with wild-type FtsZ. FtsZ has to be at or above the critical concentration for copolymerization to occur, indicating that FtsZ is nucleating the copolymers. The copolymers formed are relatively stable and appear to be stabilized by a GTP-cap. These results indicate that FtsZ2 cannot nucleate assembly in vitro, although it must in vivo. Furthermore, the stability of FtsZ-FtsZ2 copolymers argues that FtsZ2 polymers would be stable, suggesting that stable FtsZ polymers are able to support cell division.

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Figures

FIG. 1
FIG. 1
Assay of FtsZ and FtsZ2 polymerization by 90° angle light scattering. Reaction mixtures (300 μl) containing FtsZ (open circles) or FtsZ2 (open squares) at a concentration of 500 μg/ml were incubated in polymerization buffer at 30°C for 8 min, after which, 0.1 mM GTP was added to initiate polymerization. (A) The net change in light scattering following GTP addition was plotted against time. The reaction mixtures for FtsZ (B) and FtsZ2 (C) were also examined by electron microscopy at 1 min. The black bar in panel C represents 100 μm.
FIG. 2
FIG. 2
Assay of the GTPase activities of FtsZ and FtsZ2 at different protein concentrations. The GTPase activities of FtsZ and FtsZ2 were measured at different protein concentrations in polymerization buffer at 30°C with 0.2 mM [γ-32P]GTP. The specific activity was plotted against protein concentration.
FIG. 3
FIG. 3
DEAE-dextran does not affect the GTPase activities of FtsZ or FtsZ2. (A) The GTPase activities of FtsZ or FtsZ2 at a concentration of 200 μg/ml were measured in polymerization buffer at 30°C with and without 50 μg of DEAE-dextran per ml with 1 mM [γ-32P]GTP. Open circles, FtsZ; solid circles, FtsZ plus DEAE-dextran; open squares, FtsZ2; solid squares, FtsZ2 plus DEAE-dextran. (B) Assay of FtsZ2 polymerization in the presence of DEAE-dextran by electron microscopy. FtsZ2 at a concentration of 200 μg/ml was incubated in polymerization buffer at 30°C with 1 mM GTP and 50 μg of DEAE-dextran per ml. Samples were taken at 5 and 60 min for electron microscopy. Both samples were similar, so only the 60-min sample is shown. The black bar represents 100 μm.
FIG. 4
FIG. 4
Sedimentation equilibrium centrifugation of FtsZ and FtsZ2. (A) Concentration-dependent formation of FtsZ (open circles) and FtsZ2 (open triangles) oligomers is presented as the association state (Mw/M1) against cell loading concentration. (B) Isodesmic fit (solid lines) to the FtsZ distributions at an initial concentration of 10 μM (open circles) and in the presence of 4 M guanidinium hydrochloride (open triangles) are shown. The top panel shows the distribution of the residuals from each curve fit. All samples were centrifuged at 10,000 rpm for 48 h.
FIG. 5
FIG. 5
Copolymerization of FtsZ-GFP and FtsZ2 assayed by centrifugation and SDS-PAGE. Reaction mixtures (100 μl) containing 200 μg of FtsZ-GFP (lanes 1 and 2), FtsZ2 (lanes 3 and 4), or FtsZ-GFP plus FtsZ2 (lanes 5 and 6) per ml were incubated in polymerization buffer containing 1 mM MgCl2 and either 1 mM GDP (lanes 1, 3, and 5) or 1 mM GTP (lanes 2, 4, and 6). After incubation for 2 min at 30°C, the samples were centrifuged at 80,000 rpm for 15 min. The pellets were suspended in 100 μl of SDS sample buffer, and 20-μl aliquots were run on SDS-PAGE gels. The relative spot density units were for FtsZ-GFP, 19250 (lane 1) and 26100 (lane 2). The units for FtsZ2 were 8,470 (lane 3), 9,240 (lane 4), 10,010 (lane 5), and 23,100 (lane 6). Only the latter represents a significant increase over that seen in lanes 3 to 5.
FIG. 6
FIG. 6
Efficiency of assembly of FtsZ2 into the copolymers. FtsZ2 was incubated at 30°C in polymerization buffer containing 1 mM MgCl2 and1 mM GTP. After 3 min, 2 μM FtsZ-GFP (130 μg/ml) was added to each of the reaction mixtures, and incubation continued for 5 min. The reaction mixtures were centrifuged at 80,000 rpm for 15 min, the pellets were suspended in 100 μl of SDS sample buffer, and 20 μl was analyzed by SDS-PAGE. The amounts of FtsZ-GFP and FtsZ2 in the copolymers were determined by densitometry.
FIG. 7
FIG. 7
FtsZ and FtsZ2 copolymers have increased stability. (A) Various concentrations of FtsZ2 were incubated in polymerization buffer with 0.1 mM GTP at 30°C for 5 min (300 μl), and polymerization was initiated by adding FtsZ at 100 μg/ml (2.5 μM). The net change in light scattering was plotted against time. To one of the reaction mixtures was added 1 mM GDP, as indicated by the arrow. (B) Effect of varying the FtsZ2 concentration on the GTPase activity of FtsZ. The GTPase activity of FtsZ at a concentration of 100 μg/ml was measured without and with various concentrations of FtsZ2 in polymerization buffer at 30°C with 0.2 mM [γ-32P]GTP.
FIG. 8
FIG. 8
Copolymerization of FtsZ and FtsZ2 requires FtsZ to be above the critical concentration. FtsZ at 40 μg/ml (open circles) or 100 μg/ml (open squares) was added to a reaction mixture containing 0.1 mM GTP and FtsZ2 at 300 μg/ml.
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References

    1. Adams E T J, Lewis M S. Sedimentation equilibrium in reacting systems. VI. Some applications to indefinite self-associations. Studies with beta-lactoglobulin A. Biochemistry. 1968;7:1044–1053. - PubMed
    1. Bi E, Lutkenhaus J. Analysis of ftsZ mutations that confer resistance to the cell division inhibitor SulA (SfiA) J Bacteriol. 1990;172:5602–5609. - PMC - PubMed
    1. Bi E, Lutkenhaus J. Isolation and characterization of ftsZ alleles that affect septal morphology. J Bacteriol. 1992;174:5414–5423. - PMC - PubMed
    1. Bi E, Lutkenhaus J. Cell division inhibitors SulA and MinCD prevent formation of the FtsZ ring. J Bacteriol. 1993;175:1118–1125. - PMC - PubMed
    1. Bi E F, Lutkenhaus J. FtsZ ring structure associated with division in Escherichia coli. Nature. 1991;354:161–164. - PubMed

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