Functionality of proteins used in the present study.
For the in vitro studies described below, we made use of His6
-tagged versions of MinC, MinD, and MinE. Gfp-MinC-H is a MinC derivative in which residues 1 to 4 have been replaced with Gfpmut2 (6
) and the linker peptide ASMTGGQQMGRIP. In addition, the peptide LEH6
is fused to the C-terminal residue. H-MinD contains the tag MRGSH6
fused to the N-terminal starting methionine of native MinD, and MinE-H contains the tag EH6
in place of the C-terminal lysine residue of native MinE.
To ensure that these derivatives are functional in vivo, we constructed plasmids pLL19 [c
], pLL61 (Plac
), and pLL67 (Plac
). Plasmid pLL19 expresses gfp-minC-h
from the λR promoter in a temperature-dependent fashion (23
). Plasmid pLL61 encodes h-minD
and native minE
under control of the lac
promoter, and pLL67 is identical to pLL61 except that minE
is replaced with minE-h
. Plasmids pLL19 and either pLL61 or pLL67 were introduced into strain LL1 (ΔminCDE
), and transformants were grown at 37°C in the absence or presence of IPTG. In the absence of IPTG, both LL1/pLL19/pLL61 and LL1/pLL19/pLL67 cells showed the classical minicell phenotype, and Gfp-MinC-H was dispersed throughout the cytoplasm. In contrast, both strains reverted to a wild-type division pattern when grown in the presence of IPTG, and the Gfp-MinC-H fusion showed typical dynamic behavior, with average pole-to-pole oscillation cycles of 51 s (LL1/pLL19/pLL61) and 63 s (LL1/pLL19/pLL67) (Fig. and Table ). The somewhat slower oscillation observed in LL1/pLL19/pLL67 cells correlated with a somewhat less effective suppression of minicell formation, suggesting that addition of the tag to MinE reduced its cellular level and/or activity to some degree (13
). Nevertheless, all three tagged proteins were clearly functional in vivo.
FIG. 1. Pole-to-pole oscillation of Gfp-MinC-H in the presence of H-MinD and MinE-H. Fluorescence and differential interference contrast (DIC) images showing the dynamics of Gfp-MinC-H in a cell of strain LL1/pLL19/pLL67 [ΔminCDE/cI(ts) PλR:: (more ...)
In vivo functionality of His-tagged Min proteinsa
The tagged Min proteins were overexpressed in ΔminCDE
strains and purified. In addition, we purified a mutant derivative of MinE-H (*MinE-H), which lacks residues 2 to 33 corresponding to a domain required for interaction with MinD (22
Native MinD was previously shown to be a weak ATPase (7
), whose ATPase activity is stimulated by the simultaneous presence of phospholipid vesicles and MinE (19
). To ensure our preparations of H-MinD and MinE-H contained active protein, we measured ATPase activity of H-MinD in the absence or presence of MinE-H and in the absence or presence of the large phospholipid vesicles described further below (Fig. ). Addition of either lipid vesicles or MinE-H alone stimulated H-MinD-dependent ATPase activity only marginally (twofold or less). As was observed with the native forms of MinD and MinE (19
), however, the presence of both vesicles and MinE-H stimulated the ATPase activity of H-MinD almost 12-fold. Specific activities, furthermore, were similar to those reported for the native protein (7
). As expected, *MinE-H failed to stimulate the ATPase activity of H-MinD, even in the presence of lipid vesicles (Fig. ).
FIG. 2. MinD ATPase assays. (A) Stimulation of H-MinD ATPase activity by phospholipid and MinE-H. Proteins (each at 12 μM) were added to RB containing 1 mM [α-32P]ATP (0.33 μCi/nmol) and 0.5 mg of phospholipid vesicles/ml as indicated. (more ...)
Based on these results, we conclude that the tagged Min proteins retained both the physiological and biochemical activities of the native proteins.
MinD associates with phospholipid membranes in an ATP-dependent fashion.
To assess the association of purified H-MinD with phospholipids in vitro, we devised a simple and rapid phospholipid vesicle sedimentation assay. A mixture of total E. coli phospholipids containing rhodamine-labeled phosphatidylethanolamine as a tracer (0.5% of total phospholipid) was used to prepare large phospholipid vesicles. The method of preparation yielded a mixture of uni- and multilamellar vesicles of various sizes, as judged by light (see below) and electron microscopy (not shown). The majority of these vesicles were sufficiently large to be readily sedimented during a short centrifugation step.
In a typical experiment, purified H-MinD (to 2 μM) was added to RB (25 mM Tris-Cl, 50 mM KCl; pH 7.5) containing 500 μM ADP or ATP and either no lipid or 0.5 mg of phospholipid vesicles/ml. MgCl2 was added to 1 mM, and the mixtures were incubated at 30°C. After 15 min, the mixtures were subjected to centrifugation for 1 min at 16,000 × g, and aliquots of the supernatant and pellet fractions were used to determine the amounts of phospholipid and/or H-MinD in each. The results of one such experiment are shown in Fig. . Of the reactions containing ADP and ATP, respectively, 97 and 99% of phospholipid was recovered in the pellet fractions. Interestingly, 52% of H-MinD cosedimented with the vesicles in the presence of ATP. In contrast, only 4 to 7% of the protein was found in the pellet fractions of the reactions containing vesicles and ADP or containing either nucleotide but no lipid (Fig. ). This result indicated that the ATP-bound form of MinD (MinD.ATP) had a significantly higher affinity for phospholipid membrane than the ADP-bound form (MinD.ADP).
FIG. 3. ATP-dependent association of MinD with phospholipid vesicles. SYPRO Ruby-stained gel showing sedimentation of H-MinD in the presence of lipid vesicles and ATP. Purified H-MinD (to 2 μM) was added to RB containing a 500 μM concentration (more ...)
During the course of the present study, essentially the same experiment as that shown in Fig. was performed repeatedly as part of the experiments described further below. In eight separate experiments using comparable conditions (i.e., 2 μM H-MinD, 500 μM ATP or ADP, 0.5 to 1.0 mg of lipid/ml, 1 mM MgCl2), the fractions of H-MinD cosedimenting with vesicles in the presence of ADP ranged from 7 to 25%, with a mean (x) of 15.0% and a standard deviation (σ) of 5.7%, whereas in the presence of ATP the fractions ranged from 50 to 62% (x = 56.2%, σ = 3.5%). In each case, the amount of H-MinD cosedimenting with vesicles in the presence of ATP was severalfold higher (x = 4.3, σ = 1.6, range = 2.3 to 7.4) than in the presence of ADP. The variability in the amounts of H-MinD cosedimenting with vesicles in the presence of ADP suggested some aspecific binding and/or trapping of H-MinD by some batches of phospholipid vesicles, which were prepared fresh daily. To avoid this and other potential sources of nonspecific variability, care was taken throughout the present study to perform critically related sets of sedimentation assays as part of self-contained experiments, with the exact same batches of reagents.
ADP and dATP compete to a similar extent with ATP for binding to MinD (7
). To assess whether the poorly hydrolyzable ATP analogue ATPγS is also capable of interacting with H-MinD, we tested its ability to compete with [α-32
P]ATP in ATPase assays. As indicated in Fig. , ATPγS did compete with [α-32
P]ATP for interaction with H-MinD, albeit to a lesser extent than unlabeled ADP, dATP, and ATP itself. In contrast, GTP did not compete noticeably.
We then compared the association of H-MinD with membrane in the presence of ATP, ADP, dATP, ATPγS, or GTP. After incubation in the absence of lipid, a small amount of H-MinD (5 to 13%) was recovered in the pellet fraction regardless of the nucleotide used in the reaction (Table ). In the presence of lipids, this fraction increased further in all cases and also in the absence of nucleotide (not shown). Nevertheless, the fraction of H-MinD that cosedimented with lipid vesicles was again clearly dependent on the specific nucleotide present in the reaction. Thus, whereas ~25% of H-MinD was present in the pellet after incubation with ADP, dATP, or GTP, 51% of H-MinD was recovered in the pellet after incubation with ATP. After adjustment for the incomplete sedimentation of phospholipid (93%), this value increased to 55% (Table ).
We previously determined that the binding of MinD to nucleotide requires metal. Consistent with this requirement, ATP failed to stimulate association of H-MinD with the vesicles when Mg2+ was omitted from the reaction (Table ).
Interestingly, the association of H-MinD with vesicles was also stimulated by ATPγS, indicating that the binding of MinD.ATP to membrane does not require hydrolysis of the nucleotide. Although ATPγS clearly stimulated H-MinD binding to vesicles, ATPγS was consistently somewhat less effective than ATP (Table ; see also below). This may be partly or wholly due to a relatively low affinity of MinD for ATPγS, as is indicated by the nucleotide competition experiments (Fig. ).
In a recent independent study, Hu et al. (17
) similarly showed that native MinD binds phospholipid vesicles in the presence of ATP or ATPγS. Both that study and the present results support a simple model in which MinD that is bound to ATP and Mg2+
(MinD.ATP) assembles on the membrane by direct interactions with phospholipids, while the ADP-bound form (MinD.ADP) is cytoplasmic.
Cooperative binding of MinD to phospholipid vesicles.
To characterize the interaction of H-MinD with vesicles in more detail, we varied the concentrations of H-MinD, ATP, and vesicles in the mixture, as well as the time of their incubation. These experiments showed that neither the phospholipid nor the ATP concentration used in the reactions above were limiting the amount of H-MinD cosedimenting with the vesicles (results not shown). Interestingly, however, the binding of H-MinD to vesicles was markedly stimulated by increasing the concentration of the protein in the reaction.
Figure shows time course experiments in which H-MinD at various concentrations was incubated for various times with vesicles in the presence of either ATP or ADP. Within the concentration range tested (0.25 to 10.00 μM), binding of H-MinD to vesicles reached equilibrium within 1 min (excluding the 1-min centrifugation period), indicating that binding was relatively rapid.
FIG. 4. Self-enhanced association of MinD with phospholipid vesicles. (A) Time course membrane-binding assays at various H-MinD concentrations. MgCl2 (1 mM) was added to RB containing 1.0 mg of phospholipid vesicles/ml, a 500 μM concentration of either (more ...)
Interestingly, analyses of the binding isotherms showed that the binding of H-MinD to vesicles did not conform to a typical Langmuir adsorption reaction. In Fig. the fraction of bound H-MinD at equilibrium is plotted as a function of the total H-MinD concentration in the reaction. If H-MinD were to bind the phospholipid surface in a simple adsorption reaction, the amount of bound H-MinD should increase with increasing concentrations of total H-MinD, whereas the fraction of bound H-MinD should decrease (28
). In reality, however, both the amount and the fraction of H-MinD.ATP found associated with vesicles at equilibrium sharply increased with the total H-MinD concentration in the reaction. For example, whereas only ~16% of H-MinD.ATP associated with the vesicles when the protein was added to 0.25 μM, this fraction increased to ~65% when H-MinD was added to 5.0 μM (Fig. ). Thus, increased concentrations of H-MinD stimulated its binding to the vesicles, implying cooperativity in the association of the protein with the phospholipid surface.
Cooperative binding is similarly obvious from Scatchard analyses of the equilibrium binding data. As shown in Fig. , a plot of the bound to free H-MinD ratio versus bound H-MinD shows a pronounced convex curvature, a finding indicative of cooperative binding (28
At concentrations of total H-MinD of >5 μM, the fraction of bound H-MinD.ATP did not increase further but remained at ~65% (Fig. and data not shown). Based on experiments in which we measured the ability of H-MinD in supernatant fractions to bind a fresh supply of vesicles, we estimate that 10 to 15% of our purified H-MinD preparation consisted of inactive protein (not shown). Accordingly, the fraction of bound H-MinD could be increased somewhat further by enrichment for active protein in a prior round of cosedimentation with lipid vesicles. For the experiments in Table , H-MinD (5 μM) was incubated for 10 min at 30°C with vesicles (1 mg/ml) and a 100 μM concentration of either ATP or ATPγS. After sedimentation for 1 min, 66 and 51% of H-MinD, respectively, were recovered in the pellet fractions of the mixtures containing ATP and ATPγS. The pellets were resuspended in RB containing 500 μM ATP, ATPγS, or ADP. Mixtures were next incubated for 10 min at 30°C and then sedimented again (25 min at 16,000 × g) to determine the fractions of bound H-MinD. Whether vesicles had been prefractionated with H-MinD.ATP or H-MinD.ATPγS in the first round, the results were very similar. Thus, ca. 77, 60, or 22% of H-MinD cosedimented with either variety of prefractionated vesicles upon incubation with ATP, ATPγS, or ADP, respectively, in the second round (Table ). The finding that incubation with ADP led to release of H-MinD from both types of predecorated vesicles further demonstrated that binding of both H-MinD.ATP and H-MinD.ATPγS to membrane is a reversible interaction.
Effect of prior copurification with vesicles on the fraction of membrane-associating H-MinDa
MinE stimulates dissociation of MinD from phospholipid membrane.
It has been proposed that MinE stimulates dissociation of MinD from the membrane (13
). To determine the effect of the MinE protein on the MinD-phospholipid association in our in vitro system, purified H-MinD (2 μM) was incubated with nucleotide (500 μM) and phospholipid vesicles (0.5 mg/ml) as described above. After 10 min, purified MinE-H (2 μM), *MinE-H (2 μM), or MinE storage buffer was added and, after an additional 5 min, lipid vesicles and associated protein were pelleted by centrifugation for 1 min.
As in the experiments described above, only a small fraction of H-MinD (~10%) cosedimented with the vesicles when ADP was used as the nucleotide, regardless of whether or not MinE-H had been added to the reaction, and a significant fraction of H-MinD (59%) was found associated with phospholipid after incubation with ATP and when MinE-H was omitted from the reaction (Fig. ). Interestingly, however, addition of MinE-H led to a sharp reduction in the amount of H-MinD (to 19%) associated with the vesicles (Fig. ). Time course experiments showed that the fraction of bound H-MinD reached a new, and lower, plateau within 2.5 to 5 min upon addition of MinE-H to the mixtures (Fig. ).
FIG. 5. MinE-stimulated dissociation of MinD from vesicles. (A) SYPRO Ruby-stained gels showing the effect of MinE-H on the binding of H-MinD to vesicles. MgCl2 (1 mM) was added to RB containing 2 μM H-MinD, 0.5 mg of phospholipid vesicles/ml, and a 500 (more ...)
Consistent with the finding that the N-terminal domain of MinE is required for its interaction with MinD (22
), the addition of *MinE-H failed to stimulate dissociation of H-MinD from the vesicles (Fig. , 52% of H-MinD in pellet).
The same effect of MinE on MinD-membrane association was evident when MinE-H was added to vesicles that had been predecorated with H-MinD (Fig. and Table ). After incubation of predecorated vesicles with either *MinE-H or a buffer control for 5 min in the presence of ATP, ~82% of H-MinD remained associated with vesicles after sedimentation for 1 min. Upon incubation with MinE-H, however, only 50% of H-MinD still cosedimented with vesicles. This percentage decreased still further when the centrifugation step was extended from 1 to 25 min. Also, in this case more than 80% of H-MinD remained bound to membrane when incubated with buffer or *MinE-H, but only ~32% did so upon incubation with MinE-H. Both the MinE-H-stimulated dissociation of H-MinD from vesicles and the enhancing effect of an extended centrifugation period were quite reproducible (Table ), although in some experiments (e.g., Fig. ) the effects of MinE-H were more pronounced than in others. Why a longer centrifugation period resulted in a somewhat more efficient MinE-dependent dissociation of H-MinD is not clear. Time course experiments indicated that the effect is not merely due to a longer total incubation period of the reagents (Fig. and results not shown). The segregation of vesicles to the bottom of the reaction vessel may simply reduce the efficiency by which H-MinD molecules that have been released can reassociate with the vesicles, but other possibilities exist as well.
FIG. 6. Stabilization of a MinE-MinD-membrane complex in the presence of ATPγS. SYPRO Ruby-stained gels showing the effect of MinE-H on, and the interaction of MinE-H with, prefractionated H-MinD-membrane complexes are shown. (A) Vesicles were decorated (more ...)
MinE-H stimulated dissociation of H-MinD from predecorated vesiclesa
Only small amounts of the MinE-H protein (and virtually none of the *MinE-H protein) were recovered in the pellet fractions in these experiments (Fig. and Table ). Nevertheless, the amounts of MinE-H cosedimenting with the vesicles reproducibly increased with the amount of H-MinD in the pellet fractions. Thus, whereas ~4.5% of MinE-H (4.5 pmol) was recovered in the pellet, together with ~32% of H-MinD (21.4 pmol), in the 25-min centrifugation experiments, ~16% of MinE-H (16.0 pmol) cosedimented with 50% of H-MinD (33.5 pmol) in the 1-min centrifugation experiments (Table ). These results are consistent with previous work showing that, in vivo, MinE is recruited to the membrane in a MinD-dependent fashion (35
). The fact that only small amounts of MinE-H were found to cosediment with the vesicles in these experiments was also not unexpected. If MinE both requires membrane-bound MinD to associate with the vesicles itself and stimulates dissociation of MinD from the membrane, the association of MinE with MinD-decorated vesicles is expected to be short-lived because its action at the membrane will result in its own dissociation from the membrane.
Role of nucleotide hydrolysis in MinD/MinE dissociation from the membrane.
To assess the role of nucleotide hydrolysis in the MinE-stimulated dissociation of MinD.ATP from phospholipids, we compared the ability of MinE-H to dislodge H-MinD from vesicles that were decorated with either H-MinD.ATP or H-MinD.ATPγS. Decorated vesicles (at ~3.0 μM H-MinD) were incubated with the appropriate nucleotide and with various amounts of MinE-H for 5 min, and the fractions of proteins cosedimenting with vesicles were plotted as a function of MinE-H concentration. Interestingly, membrane-bound H-MinD.ATPγS proved significantly more resistant to the action of MinE-H than membrane-bound H-MinD.ATP (Fig. and ). For example, incubation of the H-MinD.ATP-decorated vesicles with 1.0 μM MinE-H reduced the fraction of bound H-MinD from a maximum of 64% (no MinE-H added) to 35%, a value close to the minimum of 28% reached after incubation with 5.0 μM MinE-H (Fig. and ). In contrast, membrane-bound H-MinD.ATPγS appeared to be impervious to MinE-H at up to 1.0 μM, and the fraction of bound H-MinD dropped only moderately from 58% (no MinE-H added) to 43% upon incubation with 5.0 μM MinE-H (Fig. and ).
FIG. 7. Recruitment of MinE to MinD-decorated vesicles. MgCl2 (1 mM) was added to phospholipid vesicles (1 mg/ml) in RB containing a 500 μM concentration of either ATP (A) or ATPγS (B), and either 5 μM H-MinD or no H-MinD. After incubation (more ...)
As described above (Table ), the fractions of cosedimenting MinE-H and H-MinD appeared to be correlated. Upon incubation of H-MinD.ATP-decorated vesicles with a low concentration of MinE-H (0.25 μM), 23% of MinE-H and 59% of H-MinD cosedimented with vesicles. However, bound fractions of both proteins dropped rapidly at higher concentrations of MinE-H. For example, when MinE-H was added to 2.5 μM, only 6% of MinE-H was recovered in the pellet, together with 34% of H-MinD (Fig. ). Interestingly, significantly higher fractions of MinE-H were retained by the vesicles decorated with H-MinD.ATPγS (Fig. and ). When it was added to 0.25 μM, 68% of MinE-H cosedimented with 63% of H-MinD, and when it was added to 2.5 μM, 21% of MinE-H cosedimented with 54% of H-MinD. Less than 10% of MinE-H sedimented when H-MinD was omitted from any of these reactions, demonstrating that the association of MinE-H with the vesicles was indeed dependent on H-MinD (Fig. and ).
In Fig. the effect of ATPγS on the binding of H-MinD and MinE-H to vesicles is displayed by plotting the ratio of bound MinE-H to bound H-MinD against the total MinE-H concentration. In the presence of ATP, this ratio already reached a small maximal value of 0.084 at 1.0 μM MinE-H. In the presence of ATPγS, however, the ratio only began to plateau at 2.5 μM MinE-H and reached a value of up to 0.36 at 5.0 μM MinE-H. From the latter value we deduce that as few as three molecules of H-MinD are sufficient to tether one molecule of MinE-H to the phospholipid surface under these conditions.
These results show that MinE-H can be directly recruited to MinD-decorated phospholipid vesicles in vitro. Furthermore, they indicate that hydrolysis of the nucleotide is required for efficient MinE-stimulated release of MinD and, hence, MinE itself, from the membrane. Similar conclusions were reached in the recent study by Hu et al. (17
MinD-dependent recruitment of the division inhibitor MinC to membrane.
MinD is thought to recruit MinC to the membrane in vivo through a direct interaction with the C-terminal D-domain of MinC (D
). To determine whether this recruitment can be mimicked in vitro, purified H-MinD (2 μM) was allowed to assemble on phospholipid vesicles (0.5 mg/ml) in the presence of ATP (500 μM) for 5 min, and purified Gfp-MinC-H was added to 0.5 μM. After an additional 10 min, mixtures were separated into soluble and insoluble fractions by centrifugation for 1 min. The amounts of H-MinD and phospholipid in each fraction were determined as described above, and the amounts of Gfp-MinC-H were determined by measuring Gfp fluorescence (Fig. ).
FIG. 8. Recruitment of MinC to MinD-decorated vesicles as shown in vesicle cosedimentation assays. The amounts of phospholipids and proteins in the pellet and supernatant fractions were determined. The values represent cosedimenting protein in percentages of (more ...)
Very little of the Gfp-MinC-H protein (<4%) was recovered in the pellet fractions when H-MinD and/or lipid vesicles were omitted from the reactions, showing that Gfp-MinC-H remained soluble in this assay system when either of these components was missing. In contrast, 86% of Gfp-MinC-H was recovered in the pellet fraction when both H-MinD and phospholipid were present in the reaction, indicating that Gfp-MinC-H bound to H-MinD-decorated vesicles (Fig. ). Time course experiments showed that the binding reaction of Gfp-MinC-H reached equilibrium within 1 min of addition of H-MinD to the mixtures (Fig. ). Thus, both the assembly of H-MinD on the vesicles and the consequent association of Gfp-MinC-H are relatively rapid processes (Fig. ).
Furthermore, titration experiments showed that H-MinD.ATP-decorated vesicles were close to being saturated with Gfp-MinC-H at the concentrations of the division inhibitor (0.5 μM) and H-MinD (2.0 μM) used in these experiments. Thus, when Gfp-MinC-H was used at 0.5 μM or above, 13 to 16 pmol of the protein cosedimented together with 35 to 41 pmol (58 to 68%) of H-MinD (Fig. ). Similar to what we deduced for the recruitment of MinE-H above, this result indicates that no more than three membrane-bound MinD monomers are sufficient to recruit one monomer of MinC.
Figure shows that Gfp-MinC-H was also efficiently recruited to vesicles decorated with H-MinD.ATPγS, indicating that hydrolysis of the nucleotide by MinD is not required for recruiting MinC to the membrane.
Recruitment of Gfp-MinC-H to H-MinD-decorated phospholipid vesicles could also be readily visualized by fluorescence microscopy. Figure show vesicles that were incubated with 2 μM H-MinD, 0.1 μM Gfp-MinC-H, and a 500 μM concentration of either ADP or ATP. In reactions containing ATP, Gfp-MinC-H was clearly seen to decorate lipid vesicles (Fig. ), whereas reactions containing ADP resulted in a homogeneous fluorescent haze in the surrounding buffer (Fig. ). As expected, similar homogenous fluorescence was observed in reactions containing ATP but lacking H-MinD (not shown).
FIG. 9. Microscopy of phospholipid vesicles showing MinD-mediated recruitment and MinE-stimulated release of MinC. MgCl2 (1 mM) was added to RB containing 2 μM H-MinD, 0.5 mg of phospholipid vesicles/ml, and 500 μM ADP (A and C) or ATP (B and (more ...)
Thus, the MinD-dependent membrane recruitment of MinC that is observed in vivo (20
) can be mimicked in vitro with a minimal set of purified components.
MinE-mediated release of both MinC and MinD from phospholipid vesicles.
The experiments above showed that H-MinD.ATP recruited both Gfp-MinC-H and MinE-H to phospholipid vesicles and that MinE-H stimulated the release of H-MinD and itself from the vesicles. In a reaction involving all three Min proteins, therefore, the MinE-stimulated release of MinD from vesicles would be expected to cause MinC to be dislodged as well. To test this, H-MinD (2 μM) was allowed to decorate vesicles for 5 min in the presence of 500 μM ATP, and Gfp-MinC-H was added to 0.1 μM. After incubation for another 5 min, either buffer or MinE-H (to 2 μM) was added, and incubation was continued for a subsequent period of 5 min. Mixtures were then fractionated by sedimentation for 1 min, and the fractions were analyzed.
As predicted, MinE-H stimulated the release of both Gfp-MinC-H and H-MinD proteins from the vesicles (Fig. ). Thus, the addition of MinE-H caused a drop in bound Gfp-MinC-H from 86 to 57% and a drop in bound H-MinD from 60 to 39%. Moreover, the MinE-H-stimulated release of both proteins was more efficient when the centrifugation step was extended from 1 to 25 min. In this case, the addition of MinE-H caused drops in bound Gfp-MinC-H and H-MinD from 85 to 13% and from 53 to 21%, respectively (Fig. ). As expected, addition of *MinE-H instead of MinE-H had no effect on the amounts of cosedimenting proteins (not shown).
FIG. 10. MinE-stimulated release of both MinC and MinD from phospholipid vesicles. MgCl2 (1 mM) was added to RB containing 2 μM H-MinD, 0.5 mg of phospholipid vesicles/ml, and 500 μM ADP or ATP. After incubation for 5 min, Gfp-MinC-H was added (more ...)
The effect of MinE-H on the association of Gfp-MinC-H with vesicles could also be monitored qualitatively by inspection of the sedimented vesicles by fluorescence microscopy. Vesicles decorated with H-MinD.ATP and Gfp-MinC-H showed a clear fluorescent signal around the vesicles, and the addition of buffer or *MinE-H prior to sedimentation had no obvious effect on this signal (Fig. ). In contrast, the addition of MinE-H markedly reduced the vesicle-associated signal to close to background (Fig. ).
MinE-mediated specific release of MinC from MinC-MinD.ATPγS-membrane complexes.
Both genetic studies in E. coli
and yeast two-hybrid assays indicated that MinE is capable of interfering with the interaction between MinC and MinD (8
). Direct evidence for such an activity of MinE came from experiments in which we compared the ability of MinE-H to stimulate the release of Gfp-MinC-H from vesicles that had been predecorated with Gfp-MinC-H and H-MinD in the presence of either ATP or ATPγS (Fig. ). Decorated vesicles were incubated with the appropriate nucleotide and various amounts of MinE-H for 5 min, and the fractions of proteins cosedimenting with vesicles were plotted as a function of MinE-H concentration (Fig. ).
FIG. 11. MinE stimulates the specific release of Gfp-MinC-H from H-MinD.ATPγS-decorated lipid vesicles. (A and B) MgCl2 (1 mM) was added to RB containing 5 μM H-MinD, 1 mg of phospholipid vesicles/ml, and 500 μM ATP (A) or ATPγS (more ...)
In the presence of ATP, MinE-H caused the release of both H-MinD and Gfp-MinC-H as described above. Release was already significant upon addition of MinE-H to 0.25 μM and was close to maximal for both H-MinD (71 to 33%) and Gfp-MinC-H (83 to 27%) when MinE-H was used at 2.5 μM (Fig. ). As in reactions lacking Gfp-MinC-H (Fig. ), H-MinD resisted release in the presence of ATPγS, and the fractions of MinE-H cosedimenting with vesicles in the presence of ATPγS were significantly greater than those in the presence of ATP at every MinE-H concentration tested (Fig. ).
In the same reactions containing ATPγS, in contrast, Gfp-MinC-H was readily released from the vesicles in a MinE-H-dependent manner. Similar to the reactions containing ATP (Fig. ), moreover, release of Gfp-MinC-H in the presence of ATPγS was close to maximal (48 to 22%) upon addition of MinE-H to 2.5 μM (Fig. ). In Fig. the relevant data are presented as plots of bound Gfp-MinC-H to bound H-MinD ratios versus the MinE-H concentration. The shape of the plots obtained with ATP and ATPγS are very similar, implying that MinE-H dislodged Gfp-MinC-H from the H-MinD.ATPγS-membrane complexes quite efficiently.
We conclude that MinE can dislodge MinC from the MinC-MinD-membrane complex, even when dissociation of MinD from the membrane is inhibited by blocking nucleotide hydrolysis.