Proper placement of the E. coli division septum requires that MinD and MinE function cooperatively to modulate the division potential of cellular sites that are located at midcell and at the cell poles. A MinD-MinE interaction in this process is implied by the fact that localization of MinE at midcell requires MinD, that MinD is required to make the MinC division inhibitor sensitive to suppression by MinE, and that MinE is required for the formation of MinD zones at the cell pole.
Gfp-MinD is capable of associating with the membrane around the entire periphery of the cell in the absence of MinE and MinC, as shown by Raskin and de Boer (16
) and as confirmed in the present study. In contrast, the membrane association of MinE (14
) and MinC (9
) both require the presence of MinD. These observations suggest that the membrane attachment of MinD is the initial step in the membrane assembly of the Min proteins. The MinD sequence does not include an apparent membrane-spanning domain, and cell fractionation and immunoelectronmicroscopic studies suggest that MinD is a peripheral membrane protein (2
). This suggests that MinD is likely to interact with another membrane component that anchors it to the membrane surface.
MinE dramatically changes the membrane distribution of MinD so that essentially all of the membrane-associated Gfp-MinD is recruited into a broad zone at one cell pole (reference 16
and this study). Previous studies with Gfp-MinE (14
) have shown that MinE forms a ring near midcell under the same conditions that lead to formation of the polar zones of MinD, raising the possibility (16
) that the midcell MinE ring plays a role in the observed redistribution of membrane-associated MinD. The present observations suggest that the two events, i.e., the formation of the midcell MinE ring and the formation of polar zones of MinD, are unrelated phenomena. Thus, in the present study the N-terminal domain of MinE, which did not form a midcell MinE ring in studies of MinE1–53
-Gfp (this study) and MinE1–33
), was capable of inducing formation of polar zones of Gfp-MinD with high efficiency. This argues against models in which the MinE ring at midcell acts as a gasket to sequester Gfp-MinD to one end of the cell and/or to provoke release of Gfp-MinD from one pole so that it can move to the opposite pole (16
fragments retain their ability to counteract the division-inhibitory action of MinCD in a MinD-dependent fashion (13
), it is likely that the N-terminal MinE domain is the domain that interacts with MinD. It is this interaction that presumably provokes the redistribution of membrane-associated MinD to the cell pole.
We suggest the following sequence of events to explain the cooperative actions of MinE and MinD, based on the idea that formation of the MinE ring at midcell and formation of the MinD zone at the cell pole both result from the lateral movement of MinD within the two-dimensional membrane matrix. First, MinD associates with the inner surface of the cytoplasmic membrane around the entire periphery of the cell. Second, the N-terminal domain of MinE interacts with the membrane-associated MinD. This recruits MinE to the membrane. We speculate that the MinD-MinE interaction may alter MinD or its membrane attachment to permit MinD molecules to diffuse laterally within the two-dimensional membrane matrix, possibly in association with its putative membrane anchor. Alternatively, MinD may always be laterally mobile within the membrane. In this case, MinE could modify MinD to increase its affinity for polar sites (discussed below). This alternative is perhaps less likely since the fact that Gfp-MinD forms polar arcs in the absence of MinE (Fig. a) implies that MinD has affinity for the cell pole independently of MinE. In either case, the laterally mobile MinD molecules are suggested to be responsible both for the formation of the MinE ring at midcell and for the formation of the MinD polar zones. Third, when the laterally mobile MinD-MinE complex encounters the putative topological target for MinE at midcell, the midcell target interacts with the C-terminal topological specificity domain of MinE, thereby anchoring MinE as a ring structure at midcell and releasing it from its MinD carrier. This finding is consistent with the observation that the topological specificity domain is required for formation of the midcell MinE ring. Fourth, unrelated to formation of the MinE ring, the collision of laterally mobile MinD molecules with a hypothetical membrane-associated nucleation site adjacent to a cell pole leads to formation of a side-by-side array of MinD molecules (the polar zone) whose assembly depends on collisional interactions within the membrane matrix. The two-dimensional MinD lattice would be expected to grow and coalesce until most or all of the mobile membrane-associated MinD molecules were captured by collision with the polar lattice. This would explain the striking observation that only a single Gfp-MinD zone was present in most cells, with no visible fluorescence elsewhere in the membrane.
The fact that the MinD zone is apparently formed at only one pole might reflect a rapid MinD assembly process following the initial interaction with one of the polar nucleation sites, a process similar to the cooperative assembly process suggested by Raskin and de Boer (16
). The forces that capture and retain MinD molecules within the polar zone have yet to be defined. The lattice could be based on direct interactions between MinD molecules or could involve the noncovalent cross-linking of MinD molecules or oligomers by another component that would also be part of the polar lattice structure. The suggested model invokes lateral diffusion of MinD molecules as the key event in the formation of the MinD polar zones and in the MinD-facilitated formation of the midcell MinE ring. The possibility also exists that the lateral translocation event might in part be an active process in which MinE modifies MinD into a form that can be actively translocated along the membrane.
A precedent for the capture of mobile membrane molecules into a single structure exists in the well-established ability of antibody molecules or lectins to induce the redistribution of eucaryotic membrane-associated surface proteins by noncovalently crosslinking them into “patches” and “caps” that are reminiscent of the Gfp-MinD structures described here (7
). Ultimately, all of the proteins are captured into a single large domain, one analogous to the Gfp-MinD zones that are formed at the cell pole.
In an entirely different type of model, MinD would move directly from the cytoplasm to the polar membrane sites after its interaction with MinE. This cannot be excluded although it would require a second mechanism to explain the requirement for MinD in formation of the midcell MinE ring. Further work will be needed to distinguish between these and other possible models.