Rod-shaped cells like E. coli
utilize the min
system to prevent the formation of polar Z rings, thereby ensuring that a Z ring only forms at midcell. This activity of the min
system is achieved by topological regulation of MinC, an inhibitor of FtsZ polymerization (8
). This inhibitor oscillates between the two halves of the cell through interaction with the MinDE oscillator (7
). For MinC to function it must contact both FtsZ and MinD. In this study we have found that these two interactions of MinC can be assigned to two functionally separable domains: an N-terminal domain which interacts with FtsZ and a C-terminal domain which interacts with MinD. Interestingly, each of these domains is also capable of mediating oligomerization.
The sequence alignments of MinCs from several bacteria (Fig. ) raised the possibility that MinC might be composed of two domains connected by a short linker. Our present studies, in which the two domains were fused to various proteins for functional and biochemical analysis, confirm this possibility. Therefore, we designate the N-terminal domain as the Z domain, since it interacts with FtsZ, and the C-terminal domain as the D domain, since it interacts with MinD.
Our results also demonstrate that MinC is an oligomer. Both the yeast two-hybrid studies and gel chromatography supported this conclusion. The gel chromatography results indicated that MalE-MinC was a dimer or possibly a trimer. We think it likely that it is a dimer and the slightly larger size estimated from the gel chromatography may be due to the shape associated with a fusion of two globular domains. The yeast two-hybrid studies also indicated that the oligomerization activity could be assigned to the D domain of MinC, and this was confirmed by fusing the D domain to MalE and demonstrating that the fusion oligomerizes. Both of these assays indicate that this oligomerization is comparable to that observed with the intact MinC. Although the yeast two-hybrid study did not indicate any self-interaction of the Z domain, surprisingly, the fusion of this domain to MalE also resulted in oligomerization. It is possible that this domain is degraded or not folded properly in yeast. The broad elution profile of MalE-MinC1–115 indicated that this domain promoted oligomers larger than the D domain and more than full-length MinC. This raises the possibility that this activity may be partially masked in the full-length MinC and exposed in the truncated MinC. It is also possible that the N-terminal domain is responsible for the larger aggregates observed during chromatography of MalE-MinC and MalE-MinC19. It is not clear what role the dimerization of MinC plays in its function.
To analyze the cell division-inhibitory activity of MinC we took advantage of the fact that overexpression of MinC, even in the absence of MinD, blocks cell division (5
). When the two domains of MinC were tested for inhibitory activity after fusion to MalE, only the Z domain was inhibitory. This domain of MinC is almost as active as the full-length MalE-MinC fusion (within two- to threefold) in both inhibiting FtsZ polymerization and inhibiting cell division. This implies that it interacts similarly with FtsZ. The assignment of FtsZ interaction to the N-terminal domain in this study is consistent with the location of the minC19
mutation. This mutation, which results in a Gly10-Asp substitution, lowers the affinity of MinC for FtsZ and its ability to interfere with FtsZ polymerization. We have also altered additional residues on either side of amino acid 10, and several of them lead to mutant proteins with reduced activity similar to that of MinC19 (Qu and Lutkenhaus, unpublished data).
Although MinC is the inhibitor of FtsZ assembly, and therefore cell division, MinD is necessary for efficient inhibition. This stimulatory effect of MinD is estimated to be 25- to 50-fold (5
) and is probably due to the MinD-dependent concentration of MinC at the membrane (7
). This recruitment is likely to involve a direct interaction between MinC and MinD, as we previously found that these proteins interact in the yeast two-hybrid system (9
). In this study we have also used this test system to demonstrate that the C-terminal domain, in addition to promoting oligomerization, is also responsible for interaction with MinD. This conclusion is also supported by the observed phenotypic effects of expressing the separate domains. The N-terminal domain inhibited division when overexpressed, but this activity was not enhanced by MinD. Also, expression of the C-terminal domain in wild-type cells caused a minicell phenotype. A competition of the D domain with full-length MinC would be expected to upset the topological regulation of division. As the concentration of the D domain rises in the cell, the MinDE oscillator would be shuffling a truncated MinC that is unable to interact with FtsZ and prevent formation of polar Z rings.
The results of the present study demonstrate the modular composition of MinC. It contains different domains that are responsible for the interaction with FtsZ and MinD. The C-terminal domain of MinC is responsible, through its interaction with MinD, for localization to the membrane and oscillation between the halves of the cell. Thus, this domain is necessary to achieve the proper topological regulation. The N-terminal domain of MinC, through its interaction with FtsZ, is responsible for its ability to inhibit division by preventing FtsZ polymerization. Thus, our results show that topological regulation of division is achieved through the fusion of a domain responsible for its localization within the cell with a domain responsible for inhibiting FtsZ polymerization.