MECHANISMS OF DIVISION SITE SELECTION AND ROLE OF THE ATP-DEPENDENT PROTEASE CLPXP IN MIND REGULATION AND PROTEOSTASIS IN \u3cem\u3eE. COLI\u3c/em\u3e

Cell division in Escherichia coli depends on mechanisms to spatially and temporally regulate selection of the division site. In dividing cells, the Z-ring, composed of linear polymers of FtsZ, assembles at the site of septation. For division to yield two identical progeny, the septum must form at the longitudinal cell center, also known as midcell. Septation at locations other than midcell results in uneven division and gives rise to daughter cells with chromosome abnormalities and functional defects, which may arise from chromosome severing or impaired chromosome segregation. Therefore, an organism\u27s overall viability necessitates cellular strategies that ensure correct placement of the Z-ring during the early stages of the division program. The Min system of E. coli, containing the proteins MinC, MinD, and MinE, represents one of the major mechanisms to spatially regulate the assembly of the Z-ring. The Min system oscillates between the poles of cell, preventing polar Z-ring assembly and thereby favoring Z-ring formation at midcell. MinC prevents assembly of the Z-ring by inhibiting FtsZ polymerization through a direct protein-protein interaction. MinD is an ATPase that associates with the membrane upon binding ATP. MinE binds to MinD to stimulate ATP hydrolysis and membrane dissociation, and therefore drives the oscillation of MinD; MinC binds MinD and is a passenger of the oscillation. In Manuscript I we review current models of how MinC disassembles FtsZ polymers and describe the molecular basis for MinDE-dependent oscillation; we further discuss recent insights into the oscillation might be regulated in vivo. In Manuscript II, we performed a mutagenesis screen to identify specific amino acid substitutions in minC conferring cell division defects to E. coli cells in vivo. We identified two distinct surface-exposed sites on MinC that are important for direct interactions with FtsZ: one on a cleft in the MinC N-domain and one on the C-domain adjacent to the MinD binding site. MinC mutant proteins that were impaired for the interaction with FtsZ, but not membrane-associated MinD, exhibited slower oscillation in vivo compared to MinC, suggesting that the FtsZ interaction with MinC modulates MinD-dependent oscillation. Furthermore, we showed that both sites on MinC identified in this study were important for the assembly of complexes between FtsZ, MinC, and membrane-associated MinD, and that the importance of each site for complex formation depended on nucleotide binding and hydrolysis by FtsZ. Importantly, we observed that the FtsZ C-terminal end (CTE), which interacts extensively with cell division proteins, was dispensable for complex assembly with dynamic FtsZ polymers formed with GTP, but required for complex assembly with FtsZ-GMPCPP and FtsZ-GDP. Many cellular processes in E. coli, including division, are regulated by degradation of key substrates. ClpXP is an ATP-dependent protease that targets substrates for irreversible proteolysis, including the E. coli cell division proteins FtsZ and ZapC; a recent study also suggested that ClpXP degrades FtsA and MinD. In Manuscript III we demonstrate that ClpXP degrades MinD in vitro and prevents copolymer assembly with MinC; furthermore, ClpXP disassembled pre-formed copolymers, which represent a class of ordered aggregates, in vitro. We quantified the rate of MinD degradation and determined that the ClpX Zinc-binding domain (ZBD) and MinD N-terminal region are both important for recognition and degradation by ClpXP. ClpXP has been reported to degrade large ordered aggregates in vitro, including FtsZ polymers and the MinCD copolymers described in Manuscript III. In E. coli, ClpXP degrades FtsZ polymers in vivo to remodel the Z-ring and accelerate polymer dynamics during constriction. In Manuscript IV, we reported that ClpXP recognizes and degrades aggregated FtsZ and Gfp appended with a ClpX recognition motif (Gfp-ssrA). Furthermore, we demonstrated that ClpX alone is capable of promoting disaggregation of heat-induced aggregates of FtsZ and Gfp, and that ClpX promotes disaggregation of FtsZ following heat shock in vivo