Abstract
Chloroplasts host photosynthesis and fulfill other metabolic functions that are essential to plant life. They have to divide by binary fission to maintain their numbers throughout cycles of cell division. Chloroplast division is achieved by a complex ring-shaped division machinery located on both the inner (stromal) and the outer (cytosolic) side of the chloroplast envelope. The inner division ring (termed the Z ring) is formed by the assembly of tubulin-like FtsZ1 and FtsZ2 proteins. ARC6 is a key chloroplast division protein that interacts with the Z ring. ARC6 spans the inner envelope membrane, is known to stabilize or maintain the Z ring, and anchors the Z ring to the inner membrane through interaction with FtsZ2. The underlying mechanism of Z ring stabilization is not well-understood. Here, biochemical and structural characterization of ARC6 was conducted using light scattering, sedimentation, and light and transmission EM. The recombinant protein was purified as a dimer. The results indicated that a truncated form of ARC6 (tARC6), representing the stromal portion of ARC6, affects FtsZ2 assembly without forming higher-order structures and exerts its effect via FtsZ2 dynamics. tARC6 prevented GDP-induced FtsZ2 disassembly and caused a significant net increase in FtsZ2 assembly when GDP was present. Single particle analysis and 3D reconstruction were performed to elucidate the structural basis of ARC6 activity. Together, the data reveal that a dimeric form of tARC6 binds to FtsZ2 filaments and does not increase FtsZ polymerization rates but rather inhibits GDP-associated FtsZ2 disassembly.
Highlights
Chloroplasts host photosynthesis and fulfill other metabolic functions that are essential to plant life
ARC6 contains several structural and functional domains depicted in Fig. 1A: chloroplast transit peptide; conserved stroma-localized N-terminal region that includes a region identified in some ARC6 homologues such as the J-like domain (89 –159); the transmembrane domain [615– 635] that is predicted to form a single helix [27]; and the conserved C-terminal domain (679 –774) located in the intermembrane space (IMS) where it interacts with chloroplast division protein PDV2 [27, 32, 36]
In all ARC6 constructs used in vitro in this study (Fig. 1A), the predicted chloroplast transit peptide that is cleaved upon import into the chloroplast as well as the nonconserved residues 68 – 88 in ARC6 have been omitted
Summary
Chloroplasts host photosynthesis and fulfill other metabolic functions that are essential to plant life. Chloroplasts are essential plant organelles that host photosynthesis and fulfill many other important metabolic functions They have to divide to maintain their numbers throughout cycles of cell division. Localization of the division site to the mid-cell in bacteria or mid-chloroplast is accomplished by proteins that inhibit Z ring assembly and/or promote its disassembly at “improper” sites. In plastids, this so-called Min system is composed of ARC3, a key negative regulator of FtsZ assembly (20 –22), and additional proteins that are believed to modulate its activity, such as MinD [23], MinE [24], MCD1 [25], and PARC6 [26, 27].
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