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ARC6 is a J-domain plastid division protein and an evolutionary descendant of the cyanobacterial cell division protein Ftn2 - PubMed

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Comparative Study

. 2003 Aug;15(8):1918-33.

doi: 10.1105/tpc.013292.

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Comparative Study

ARC6 is a J-domain plastid division protein and an evolutionary descendant of the cyanobacterial cell division protein Ftn2

Stanislav Vitha et al. Plant Cell. 2003 Aug.

Abstract

Replication of chloroplasts is essential for achieving and maintaining optimal plastid numbers in plant cells. The plastid division machinery contains components of both endosymbiotic and host cell origin, but little is known about the regulation and molecular mechanisms that govern the division process. The Arabidopsis mutant arc6 is defective in plastid division, and its leaf mesophyll cells contain only one or two grossly enlarged chloroplasts. We show here that arc6 chloroplasts also exhibit abnormal localization of the key plastid division proteins FtsZ1 and FtsZ2. Whereas in wild-type plants, the FtsZ proteins assemble into a ring at the plastid division site, chloroplasts in the arc6 mutant contain numerous short, disorganized FtsZ filament fragments. We identified the mutation in arc6 and show that the ARC6 gene encodes a chloroplast-targeted DnaJ-like protein localized to the plastid envelope membrane. An ARC6-green fluorescent protein fusion protein was localized to a ring at the center of the chloroplasts and rescued the chloroplast division defect in the arc6 mutant. The ARC6 gene product is related closely to Ftn2, a prokaryotic cell division protein unique to cyanobacteria. Based on the FtsZ filament morphology observed in the arc6 mutant and in plants that overexpress ARC6, we hypothesize that ARC6 functions in the assembly and/or stabilization of the plastid-dividing FtsZ ring. We also analyzed FtsZ localization patterns in transgenic plants in which plastid division was blocked by altered expression of the division site-determining factor AtMinD. Our results indicate that MinD and ARC6 act in opposite directions: ARC6 promotes and MinD inhibits FtsZ filament formation in the chloroplast.

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Figures

Figure 1.
Figure 1.

Structures of the Arabidopsis ARC6 Gene and the Encoded Protein. (A) Genetic structure. Exons are depicted as black rectangles; ATG and TAA are the translation initiation and termination codons, respectively. The nucleotide sequence flanking the mutation in arc6 at position +1141 (arrowhead) and the C-to-T transition in codon 325, which introduced a premature stop codon (TGA), are shown. nt, nucleotide; bp, base pairs. (B) ARC6 protein structure. Putative functional domains are depicted as rectangles, and their positions within the amino acid sequence are indicated: the chloroplast targeting signal (CT; vertically hatched box); the J domain (horizontally hatched box); and the transmembrane domain (TM; white box). Black lines above the diagram delineate the N- and C-terminal regions conserved among the ARC6-like proteins in plants and cyanobacteria. aa, amino acids.

Figure 2.
Figure 2.

Complementation of the arc6 Mutant Phenotype. Chloroplasts in leaf mesophyll cells. Bar = 20 μm. (A) Wild-type Ws. (B) arc6-1 mutant. (C) and (D) arc6-1 mutant transformed with the wild-type ARC6 transgene, showing full (C) or partial (D) complementation based on chloroplast size and number.

Figure 3.
Figure 3.

Sequence Alignment of J Domains. J domains from E. coli DnaJ and Hsc56 (DnaJ homolog) and from plant and cyanobacterial ARC6-like proteins were aligned with the consensus sequence (Hennessy et al., 2000). Light-gray shading indicates 70% similarity, and black shading indicates 70% identity among all J domains in the Pfam database. Protein accession numbers, where available, and organism names are listed in Table 2. Symbols above the consensus indicate residues believed to be important, in E. coli DnaJ, for maintaining J-domain structure (x), for binding to Hsp70 chaperones (#), and for the specificity of this interaction (*) (Hennessy et al., 2000).

Figure 4.
Figure 4.

Immunofluorescence and Immunoblot Analyses of FtsZ. (A) to (F) Localization of FtsZ2 in leaf mesophyll chloroplasts. Similar localization patterns also were obtained for FtsZ1 (data not shown). The immunofluorescence micrographs are shown at left, and the chloroplast shapes are drawn at right. (A) Wild-type (WT) chloroplasts, each with a single FtsZ ring (arrowheads). (B) A single, enlarged arc6 mutant chloroplast. (C) Chloroplasts from an arc6 mutant plant complemented with a wild-type copy of the ARC6 gene. Partial and complete FtsZ rings are indicated by arrowheads. (D) A single, enlarged chloroplast of an ARC6-overexpressing plant. (E) Chloroplast from an AtMinD-overexpressing plant. (F) Chloroplast from an AtMinD antisense plant. Arrowheads indicate multiple FtsZ rings in the enlarged chloroplast. Bar = 10 μm. (G) Immunoblots of leaf extracts from wild-type Ws plants (lanes 1 and 2), arc6 mutant plants (lanes 3 and 4), arc6 mutant plants complemented with ARC6 (lanes 5 and 6), ARC6-overexpressing plants (lanes 7 and 8), AtMinD-overexpressing plants (lanes 9 and 10), and AtMinD antisense plants (lanes 11 and 12) probed with antibodies specific for AtFtsZ1 (Z1) or AtFtsZ2 (Z2). The identities of the immunoreactive bands are indicated at left, and their approximate molecular masses are indicated at right. Extracts from 1 mg of fresh leaf tissue were used in each lane. FtsZ levels in wild-type Columbia plants (data not shown) were identical to those in wild-type Ws (lanes 1 and 2).

Figure 5.
Figure 5.

AtMinD RNA Analysis. (A) Ethidium bromide–stained gel with RT-PCR products amplified with primers specific for AtMinD or 18S rRNA. The number of amplification cycles and the identities of the amplified target are indicated at top, and the sources of RNA samples are shown at left. WT, wild type. (B) Quantification of RT-PCR products based on fluorescence intensities of the bands shown in (A). The band intensity of the AtMinD PCR product is expressed relative to that of the 18S rRNA after 15 cycles of amplification.

Figure 6.
Figure 6.

Chloroplast Import Assay. In vitro–synthesized, radiolabeled proteins (lane 1) were incubated with isolated pea chloroplasts. Chloroplasts then were incubated without (−, lanes 2 and 3) or with (+, lanes 4 and 5) the protease thermolysin for 30 min on ice and then quenched. After protease treatments, intact chloroplasts were recovered, lysed, and separated by centrifugation into total membrane (P) and soluble (S) fractions. Precursor protein (p) and mature protein (m) are indicated by arrows at right, and the positions of molecular mass markers are shown at left. TP, 10% of the radiolabeled translation product used in the import reaction (lane 1). (A) Arabidopsis full-length ARC6. (B) Truncated ARC6 (ARC6-572), representing the first 572 amino acids of the full-length protein. (C) Truncated inner membrane–localized Tic110-110N, facing the stromal compartment (Jackson et al., 1998). (D) Soluble, stroma-localized small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase.

Figure 7.
Figure 7.

Membrane Topology of Arabidopsis ARC6. (A) After the in vitro import of radiolabeled ARC6 (lanes 2 and 3), pea chloroplasts were incubated with (+) or without (−) the protease trypsin (lanes 4 to 7) or thermolysin (lanes 8 to 11). As a control for protease activity, the treatments were preformed in the presence (+) of Triton X-100 (lanes 4, 5, 8, and 9). After protease treatments, intact chloroplasts were recovered, except from the Triton X-100–treated samples, and separated by centrifugation into total membrane (P) and soluble (S) fractions. Mature protein (mARC6) is indicated by an arrowhead at right, and the positions of molecular mass markers are shown at left. The arrow in lane 6 indicates the position of a truncated ARC6 protein band resulting from trypsin treatment of intact chloroplasts. The lower molecular mass bands, unrelated to protease treatment, are indicated with asterisks in lanes 1, 2, and 10. TP, 10% of the radiolabeled translation product used in the import reaction (lane 1). (B) Proposed membrane topology of ARC6 in chloroplasts. The C and N termini of ARC6 are labeled as C and N, respectively. The accessibility of the outer, cytosolic chloroplast surface of and the envelope intermembrane space to proteases is indicated by arrows. OM and IM, outer and inner envelope membranes, respectively.

Figure 8.
Figure 8.

Localization of an ARC6-GFP Fusion Protein in Transgenic Arabidopsis Plants, and Rescue of the arc6 Mutant Phenotype by the Fusion Protein. Arrows indicate concentrated areas of GFP fluorescence. The shapes of the chloroplasts are apparent from the dim background fluorescence. Single arrowheads indicate ARC6-GFP localized to the plastid midpoint. The double arrowhead in (B) indicates an additional ARC6-GFP strand not associated with the midpoint. Bars = 5 μm. (A) and (B) GFP fluorescence in leaf mesophyll cell chloroplasts from two independent T1 plants expressing the ARC-GFP transgene in a wild-type (Columbia) background. A similar localization pattern was observed in the wild-type Ws background (data not shown). (C) GFP fluorescence in a cotyledon mesophyll cell from a 6-day-old T1 plant expressing the ARC-GFP transgene in the arc6 mutant background. The presence of multiple chloroplasts in cotyledons and in true leaves (data not shown) indicates complementation of the arc6 chloroplast division defect by the fusion protein.

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References

    1. Addinall, S.G., and Holland, B. (2002). The tubulin ancestor, FtsZ, draughtsman, designer and driving force for bacterial cytokinesis. J. Mol. Biol. 318, 219–236. - PubMed
    1. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. (1990). Basic local alignment search tool. J. Mol. Biol. 215, 403–410. - PubMed
    1. Bi, E., and Lutkenhaus, J. (1991). FtsZ ring structure associated with division in Escherichia coli. Nature 354, 161–164. - PubMed
    1. Bi, E., and Lutkenhaus, J. (1993). Cell division inhibitors SulA and MinCD prevent formation of the FtsZ ring. J. Bacteriol. 175, 1118–1125. - PMC - PubMed
    1. Bramhill, D. (1997). Bacterial cell division. Annu. Rev. Cell Dev. Biol. 13, 395–424. - PubMed

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