| Chloroplasts are specialized organelles evolved from cyanobacteria as endosymbionts in plant cells. Homologs of the cyanobacterial cell division genes FtsZ, MinD and MinE have been identified previously in plants and shown to be important for chloroplast division. In this work, a new chloroplast division gene, ARC5, was identified by positional cloning in Arabidopsis. The arc5 mutant has 1--15 enlarged dumbbell-shaped chloroplasts per cell, suggesting that ARC5 may be involved in the constriction of chloroplasts during division. A GFP-ARC5 fusion protein rescues the mutant phenotype of arc5 and localizes to a ring at the chloroplast division site. Chloroplast import and protease protection assays indicate that the ARC5 ring is positioned on the outer surface of the chloroplast. Thus, ARC5 is the first cytosolic component of the chloroplast division complex to be identified. Phylogenetic analysis suggests that ARC5 is in the dynamin family of mechanochemical enzymes in eukaryotes, which are large GTPases involved in membrane fission and fusion in a variety of cellular processes. These results indicate that the chloroplast division machinery is of mixed evolutionary origin and that it shares structural and mechanistic similarities with both the cell division machinery of bacteria and the dynamin-mediated organellar fission machineries of eukaryotes. Additional experimental results demonstrate that a homolog of ARC5, ARC5H, is not involved in chloroplast division. Therefore, ARC5 is important but not essential for chloroplast division and it probably evolved to facilitate the division of chloroplasts during endosymbiosis.; FZO is a more diverged dynamin-related protein involved in mitochondrial fusion in metazoa and fungi. However, an FZO-like protein in Arabidopsis , FZL, is targeted to chloroplasts. fzl knockout mutants show a chloroplast division defect with heterogeneity in chloroplast size and thinner thylakoid sacs with disorganized stacking. An FZL-GFP gene rescued the fzl mutant phenotype. Overexpression of FZL-GFP causes very long thylakoid sacs with considerably less stacking. FZL-GFP is a membrane protein present in both the thylakoids and chloroplast inner membrane. FZL-GFP is mainly localized to punctate or vesicle-like structures associated with chloroplasts and infrequently in the cytosol, but is not associated with mitochondria. The level of FZL-GFP expression correlates with the number of punctate and vesicle-like structures on the surface of the chloroplast, suggesting that these structures are coincident with FZL and that FZL may be involved in their formation. Mutation of a conserved lysine residue in the GTPase domain abolishes the punctate localization pattern and the ability of FZL-GFP to complement fzl mutant. FZL seems to be involved in the formation and trafficking of vesicles from the thylakoid to the chloroplast envelope and cytosol, and blocking of this process affects both thylakoid morphology and chloroplast division.; The finding that two dynamin-related proteins of eukaryotic origin, ARC5 and FZL, are required for chloroplast division and morphogenesis indicates that the chloroplast division machinery has been modified from the cyanobacterial cell division machinery during evolution of the organelle. |
