The switch I and II regions of MinD are required for binding and activating MinC

Abstract

MinD and MinC cooperate to form an efficient inhibitor of Z-ring formation that is spatially regulated by MinE. MinD activates MinC by recruiting it to the membrane and targeting it to a septal component. To better understand this activation, we have isolated loss-of-function mutations in minD and carried out site-directed mutagenesis. Many of these mutations block MinC-MinD interaction; however, they also prevent MinD self-interaction and membrane binding, suggesting that they affect nucleotide interaction or protein folding. Two mutations in the switch I region (MinD box) and one mutation in the switch II region had little affect on most MinD functions, such as MinD self-interaction, membrane binding, and MinE stimulation; however, they did eliminate MinD-MinC interaction. Two additional mutations in the switch II region did not affect MinC binding. Further study revealed that one of these allowed the MinCD complex to target to the septum but was still deficient in blocking division. These results indicate that the switch I and II regions of MinD are required for interaction with MinC but not MinE and that the switch II region has a role in activating MinC.

FIG. 1.

Location of mutations in minD. This diagram indicates the conserved motifs that have been identified within MinD. The location of the mutations and the amino acid substitutions that were analyzed in this study are indicated. The mutations above the diagram were obtained by random mutagenesis and screening for loss of MinD function. The mutations below the diagram were constructed by site-directed mutagenesis.

FIG. 2.

Effect of minD mutations on membrane localization. N-terminal GFP fusions to the various MinD mutant proteins were examined in JS964 (Δmin). Cells containing the fusions were fixed with 2% glutaraldehyde 1 h after induction of the fusion with 0.001% arabinose. Cells were analyzed by fluorescence microscopy. (A) Wild type; (B) MinD-R44G; (C) MinD-G42A; (D) MinD-S121T; (E) MinD-I141N.

FIG. 3.

ATPase activity of MinD mutants. MinD (D) proteins (9 μM) were mixed with phospholipid (PL) vesicles (400 μg/ml) and analyzed for ATPase activity in the presence or absence of MinE (E) (9 μM) as indicated.

FIG. 4.

Interaction of MinD mutants with phospholipid vesicles. (A) MinD switch I mutants bind to phospholipid vesicles. MinD proteins (4 μM) were mixed with 400 μg of phospholipid vesicles/ml in the presence of 1 mM ATP. After adding the ATP, the samples were centrifuged and the pellets were analyzed by SDS-PAGE. (B) MinE stimulates release of MinD switch I mutants from vesicles. MinD proteins were incubated with phospholipid vesicles as described for panel A in the presence (+) or absence (−) of MinE (4 μM). (C) Analysis of the ability of switch I and switch II mutants to recruit MinC to vesicles. MinD (4 μM) was incubated with phospholipid vesicles in the presence of 1 mM ADP or ATP. MalE-MinC^116-231 (4 μM) was added, the samples were centrifuged, and the pellets were analyzed by SDS-PAGE.

FIG. 5.

Switch II mutants localize to the membrane in vivo. GFP fusions to the various MinD switch II mutants were analyzed for membrane localization as described in the legend to Fig. 2. (A) MinD- IE125,126AA; (B) MinD-E126A; (C) MinD-I125E.

FIG. 6.

The switch II mutant MinD-E126A targets MinC^116-231 to septal rings. The ability of the MinD-E126A mutant to target MinC to the septal machinery was assessed by fluorescence microscopy. JS964 (Δmin) containing the control plasmid pHJZ109 (gfp-minC^116-231-minD) (A) or pHJZ109-D9 (gfp-minC^116-231-minD-E126A) was grown in Luria-Bertani broth at 37°C until the optical density at 600 nm reached 0.05. IPTG was added at 40 μM, and the cells were fixed with 2% glutaraldehyde 1 h later. Cells were analyzed by fluorescence microscopy.

FIG. 7.

MinD-E126A is unable to provide MinD function to spatially regulate division. To verify that minD-E126A lacked the ability to spatially regulate division, it was placed in the context of the min operon on a single-copy plasmid and introduced into JS964 (Δmin). Phase-contrast microscopy of exponentially growing cells is shown. (A) JS964 (Δmin) pSEB12 (minCDE); (B) JS964 (Δmin) pSEB12-D9 (minC minD-E126A minE).

FIG. 8.

Model of MinD indicating the locations of the residues investigated in this study. The MinD protein sequence of E. coli was modeled on the structure of the MinD-like protein of P. furiosus (PDB accession no. 1G3R). The C-terminal 25 residues are not in the structure, since the corresponding region is not present in the MinD-like protein of P. furiosus. The positions of the residues altered in this study are indicated. The switch I residues are colored red, and the switch II residues are colored cyan.