Chloroplasts replicate by binary fission, a process driven by ring-like dynamic division machinery at mid-chloroplast. In Arabidopsis thaliana, the first molecular assembly of this machinery, the Z-ring, forms via the association of FtsZ1 and FtsZ2 heteropolymers with the inner envelope membrane through the membrane-tethering protein ACCUMULATION AND REPLICATION OF CHLOROPLASTS6 (ARC6). Spatial control of Z-ring assembly ensures the correct placement of the division machinery and, therefore, symmetric chloroplast division. The plant-specific protein MULTIPLE CHLOROPLAST DIVISION SITE1 (MCD1) plays a role in Z-ring positioning and chloroplast division site placement, but its mechanism of action is unknown. Here, we provide evidence that MCD1 is a bitopic inner membrane protein whose C terminus faces the chloroplast stroma. Interaction analysis showed that MCD1 and ARC6 directly interact in the stroma and that MCD1 binds to FtsZ2 in an ARC6-dependent manner. These results are consistent with the in vivo observation that ARC6 influences the localization of MCD1 to membrane-tethered FtsZ filaments. Additionally, we found that MCD1 is required for the regulation of Z-ring positioning by ARC3 and MinE1, two components of the chloroplast Min (minicell) system, which negatively regulates Z-ring placement. Together, our findings indicate that MCD1 is part of the chloroplast Min system that recognizes membrane-tethered FtsZ filaments during chloroplast division-ring positioning.
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