ARC5, a cytosolic dynamin-like protein from plants, is part of the chloroplast division machinery

Abstract

Chloroplast division in plant cells is orchestrated by a complex macromolecular machine with components positioned on both the inner and outer envelope surfaces. The only plastid division proteins identified to date are of endosymbiotic origin and are localized inside the organelle. Employing positional cloning methods in Arabidopsis in conjunction with a novel strategy for pinpointing the mutant locus, we have identified a gene encoding a new chloroplast division protein, ARC5. Mutants of ARC5 exhibit defects in chloroplast constriction, have enlarged, dumbbell-shaped chloroplasts, and are rescued by a wild-type copy of ARC5. The ARC5 gene product shares similarity with the dynamin family of GTPases, which mediate endocytosis, mitochondrial division, and other organellar fission and fusion events in eukaryotes. Phylogenetic analysis showed that ARC5 is related to a group of dynamin-like proteins unique to plants. A GFP-ARC5 fusion protein 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. ARC5 has no obvious counterparts in prokaryotes, suggesting that it evolved from a dynamin-related protein present in the eukaryotic ancestor of plants. These results indicate that the chloroplast division apparatus 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.

Figure 1

Comparison of chloroplasts in Arabidopsis leaf mesophyll cells. (A) Wild-type (Ler). (B and C) arc5. Cells are from fixed tissue. (Bars, 10 μm.)

Figure 2

Cloning of ARC5. (A) Fine mapping of ARC5. Triangle indicates the position of ARC5. (B) ORFs of ARC5. Black boxes represent exons; solid lines represent introns; gray box indicates the alternatively spliced intron. The mutation in arc5 and its position are indicated. (C) Amplification of the transgene inserts in two arc5-like T₁ plants produced by transforming wild-type plants (Col-0) with a library of sense and antisense fragments derived from MMB12 and MPN9. A 100-bp DNA ladder (New England Biolabs) is shown at left. Arrow indicates a HindIII–HincII fragment from At3g19730 common to both arc5-like transgenic lines. (D–G) Single leaf mesophyll cells from Col-0 wild-type (D), an arc5-like T₁ plant described in C (E), the arc5 (Ler) parent line used for complementation by ARC5 (F), and an arc5 plant transformed with the ARC5 gene (G). (Bars in D–G, 10 μm.)

Figure 3

ARC5 is a dynamin-like protein. (A) Alignment of ARC5 with Dynamin-1 from Homo sapiens and Dnm1p from Saccharomyces cerevisiae. Gray boxes indicate completely conserved residues; yellow boxes are identical residues; cyan boxes are similar residues; dashes indicate gaps. The domain structure is indicated by the lines above the alignment. Red, GTPase domain; green, middle domain; blue, PH domain; lavender, GTPase effector domain; black, PR domain. The dotted underline indicates the sequence encoded by the alternatively spliced intron in ARC5. The triangle indicates the position of the arc5 mutation. (B) Phylogenetic analysis of ARC5 with an unrooted neighbor-joining tree. Bootstrap values are shown at selected nodes. The first and second bootstrap values are from the neighbor-joining and parsimony analyses, respectively. Nodes with <50% bootstrap support, or not present, are represented by a “−”. Accession numbers for the sequences aligned with ARC5 are listed in Materials and Methods.

Figure 4

GFP–ARC5 is localized to the constriction site of dividing chloroplasts. (A) Bright-field. (B) GFP fluorescence. Arrows indicate corresponding positions in the two images. (Bars, 10 μm.)

Figure 5

ARC5 is on the outside surface of chloroplast. Radiolabeled ARC5 (Top), tp110–110N, the precursor of the inner envelope marker protein 110N (Middle), and pSS, the precursor of the stromal marker protein mSS (Bottom), were produced by coupled in vitro transcription/translation and subsequently incubated with isolated pea chloroplasts (7). Chloroplasts were recovered by centrifugation and incubated with (+) or without (−) thermolysin. Intact chloroplasts were again recovered and fractionated into membrane (P) and soluble (S) fractions. TP, translation product.