Chloroplast Division Protein ARC3: Effects on FtsZ2 Assembly and GTPase Activity

Abstract

Chloroplasts evolved from cyanobacterial endosymbiotic ancestors and their division is a complex process initiated by assembly of cytoskeletal FtsZ proteins into a ring structure at the division site (Z-ring). The cyanobacterial Z-ring positioning system (MinCDE proteins) is also conserved in chloroplasts except that MinC was lost and replaced by the eukaryotic ARC3. Both MinC and ARC3 act as negative regulators of FtsZ assembly, but ARC3 bears little sequence similarity with MinC. Here, light scattering assays, co-sedimentation, light microscopy, GTPase assay and transmission electron microscopy in conjunction with single particle analysis have been used to elucidate the structure of ARC3 and its effect on its main target in chloroplast division: FtsZ2.

Analysis of FtsZ2 in vitro assembly reactions in the presence and absence of GMPCPP showed that ARC3 promotes FtsZ2 debundling and disassembly of existing filaments in a concentration-dependent manner and requires GTP hydrolysis. 3D reconstruction of ARC3 revealed an almost circular molecule in which the FtsZ-binding N-terminus and the C-terminal PARC6-binding MORN domain are in close proximity and suggests a model for PARC6-enabled binding of ARC3 to FtsZ2. The latter is corroborated by in vivo data.