Figure 1.
The FtsZ C-terminus is important for ClpXP degradation.
A. Linear schematic diagram of FtsZ protein separated into three regions: the polymerization domain (amino acids 1 through 316), the unstructured linker (amino acids 317 through 369) and the C-terminal domain or conserved core region (amino acids 370 through 383). The C-terminal FtsZ deletions and substitution mutations used here are presented. B. Comparison of rates of degradation of FtsZ wild type and mutant proteins in the presence and absence of GTP from in vitro degradation reactions containing 10 µM wild type or mutant fluorescent FtsZ and 1 µM ClpXP. C. Structural model of the C-terminal alpha-helical region of FtsZ, residues 367 through 383, that cocrystallized with ZipA (PDB entry 1F47) [32]. Side chains are shown for R379 (red) and K380 (blue). D. Alignment of the C-terminal amino acid sequences of several proteins recognized by ClpX. C-terminal sequences shown belong to the consensus C motif-2 family of ClpX recognition tags (R/H-x-K/R-K-Φ with x representing any amino acid and Φ representing a hydrophobic amino acid residue) [21]. In B, data from 3 replicates are presented as mean ± SEM.
Figure 2.
Residues in the linker region of FtsZ are important for degradation by ClpXP.
A. Alignment of FtsZ residues 349 through 358 from the linker domain with the C-terminal ClpX recognition region of Mu repressor protein from phage Mu. B. Linear schematic diagram of FtsZ showing regions of the linker that were tested by triple alanine scanning mutagenesis of wild type FtsZ and truncated FtsZ(Δ375-383). C. Comparison of rates of degradation of wild type and mutant FtsZ, proteins in the presence and absence of GTP from in vitro degradation reactions containing 10 µM wild type or mutant fluorescent FtsZ and 1 µM ClpXP. Data from 3 replicates are presented as mean ± SEM.
Figure 3.
FtsZ mutant proteins with C-terminal mutations hydrolyze GTP and assemble into polymers.
(A) Rates of GTP hydrolysis were measured in reactions containing FtsZ wild type or mutant (5 µM) and GTP (1 mM) as described in Experimental Procedures. Data from 3 replicates are presented as mean ± SEM. (B) GTP-dependent assembly of FtsZ wild type and mutant proteins (8 µM) was monitored by 90° light scattering as described in Experimental Procedures. A baseline was collected for 2 min, then GTP was added when indicated, to stimulate polymerization. Light scattering was measured for 25 min. Data shown is representative of 3 replicates.
Figure 4.
A peptide corresponding to the C-terminus of the SspB adaptor inhibits FtsZ degradation.
A. Relative rate of FtsZ degradation by ClpXP in the presence and absence of GTP with increasing concentration of SspB peptide (10, 20, 40 or 80 µM). Relative rate of FtsZ degradation was defined by V/Vo, where V is equal to the rate in the presence of SspB peptide and Vo is equal the rate in the absence of SspB peptide. Degradation reactions contained 15 µM fluorescent FtsZ, 0.75 µM ClpXP, ATP and, where indicated, GTP. Data were fit to a nonlinear dose-response inhibitor curve. B. Comparison of relative rates of degradation of wild type FtsZ, FtsZ(Δ375-383) and FtsZ(352AAA) in the presence of 0, 20 or 50 µM SspB peptide. Reactions contained 10 µM FtsZ wild type or mutant protein, 0.75 µM ClpXP with GTP and ATP. In A and B data from 3 replicates are presented as mean ± SEM.
Figure 5.
Association of FtsZ wild type and mutant polymers with ClpX.
The interaction between ClpX and FtsZ was measured by monitoring the fraction of ClpX ATP hydrolysis mutant, ClpX(E185Q), that co-pellets with FtsZ wild type or mutant protein in the presence of GTP. Pelleted ClpX(E185Q) and FtsZ was quantified by Coomassie staining of SDS-PAGE gels and densitometry. Data from at least 3 replicates are presented as mean ± SEM.
Figure 6.
MinC competes with ClpXP for FtsZ in vitro.
A. MinC was included in degradation reactions containing FtsZ with GTP and ClpXP (0.5 µM). B. FtsZ (5 µM; 125 pmol/reaction) was first preincubated with GTP (2 mM) and then incubated with MinC (2 µM), ClpXP (0.5 µM) and ATP, as indicated. FtsZ polymer disruption was monitored by measuring the amount of fluorescent FtsZ present in high-speed centrifugation pellets. In A and B data from 3 replicates are presented as mean ± SEM.
Figure 7.
Mutations in the FtsZ C-terminal domain impair FtsZ function in vivo.
FtsZ mutant proteins were tested for function in vivo by comparing CFUs of ftsZ84 cells expressing FtsZ mutant proteins after incubation in liquid culture at the restrictive temperature (42°C) for 4 hours as described in Experimental Procedures. Data from 3 replicates are presented as mean ± SEM.