A dynamin-like protein (ADL2b), rather than FtsZ, is involved in Arabidopsis mitochondrial division

Abstract

Recently, the FtsZ protein, which is known as a key component in bacterial cell division, was reported to be involved in mitochondrial division in algae. In yeast and animals, however, mitochondrial fission depends on the dynamin-like proteins Dnm1p and Drp1, respectively, whereas in green plants, no potential mitochondrial division genes have been identified. BLAST searches of the nuclear and mitochondrial genome sequences of Arabidopsis thaliana did not find any obvious homologue of the alpha-proteobacterial-type ftsZ genes. To determine whether mitochondrial division of higher plants depends on a dynamin-like protein, we cloned a cDNA for ADL2b, an Arabidopsis homologue of Dnm1p, and tested its subcellular localization and its dominant-negative effect on mitochondrial division. The fusion protein of green fluorescent protein and ADL2b was observed as punctate structures localized at the tips and at the constriction sites of mitochondria in live plant cells. Cells expressing dominant-negative mutant ADL2b proteins (K56A and T77F) showed a significant fusion, aggregation, and/or tubulation of mitochondria. We propose that mitochondrial division in higher plants is conducted by dynamin-like proteins similar to ADL2b in Arabidopsis. The evolutional points of loss of mitochondrial FtsZ and the functional acquisition of dynamin-like proteins in mitochondrial division are discussed.

Figure 1

ADL2b is likely a member of the mitochondrial dividing dynamin-like protein family. Phylogenetic tree of dynamin and its related GTPase superfamily is shown. ADL2b is subgrouped with the yeast Dnm1p, nematode Drp1, and human Drp1 (gray ovals), all of which are mitochondria-dividing dynamin-like proteins. GenBank accession nos. for the proteins used in this analysis are as follows: WormDrp1, AF166274; HumanDrp1, AF000430; YeastDNM1, L40588; ADL1, L36939; Phragmoplastin, U25547; YeastMGM1, X62834; HumanOPA1, AB011139; ADL3, AB026987; ADL6, AF180732; WormDyn1, AF167982; FlyShibire, X59435; HumanDNM2, NM004945; Human DNM1, NM004408; and YeastVPS1, M33315. This phylogenetic tree was calculated by the N-J method (19). The bootstrap values based on 1,000 replications are shown.

Figure 2

Localization of GFP-ADL2b with tips and constriction sites of mitochondria. BY-2 cells transiently expressing GFP-ADL2b with mitochondrial marker MitoTracker were examined by confocal laser scanning microscopy. High-magnification images of a part of a single BY-2 cell are shown. Left and Center are separate images obtained with the GFP and MitoTracker of Right images, respectively. GFP-ADL2b (green) was located in small punctate structures, many of which were localized in tips and constricted sites of mitochondria (red). GFP-ADL2b spots (arrowheads) were localized to places where the mitochondria were constricted. GFP signals (arrows) were localized at the tips of mitochondria. [Bar = 5 μm.]

Figure 3

Schematic drawings of the plasmids and G1 and G2 motifs of dynamin-like proteins. (A) Schematic drawings of pSA2b (the top construct, see Materials and Methods) and the plasmids used to transform plant cells in Fig. 4. The ORFs of GFP (S65T) cDNA (GFP), its in-frame fusions with mitochondrial targeting presequence of ATPase delta subunit cDNA (Mt), and ADL2b wild type (WT), ADL2b K56A mutant (K56A), or ADL2b T77A mutant (T77A) cDNA were transiently coexpressed under the control of the CaMV 35S promoter (35S) and the terminator of nopaline synthase (^TNOS). (B) G1 and G2 motifs, the targeted sites of the in vitro mutagenesis, in the GTPase domains of representative dynamin family members. Alignment of a part of short GTPase core segment from Arabidopsis ADL2a and ADL2b, yeast (S. cerevisiae) DNM1, human Drp1, nematode (C. elegans) Drp1, and yeast (S. cerevisiae) Vps1. Underlined segments show G1 and G2 motifs in the GTPase domains of dynamin family members (22). The site-directed mutated amino acids are shown at the top. Alignment was performed by using the CLUSTAL W multiple alignment program (18). See legend of Fig. 1 for accession nos.

Figure 4

Mutagenized ADL2b causes dominant-negative effects on mitochondrial morphology. Live plant cells transiently expressing wild-type ADL2b or mutagenized ADL2b as well as Mt-GFP were examined by confocal laser scanning microscopy. (Left) Arabidopsis leaf epidermal cells expressing the vectors shown at the right end. The mitochondria were visualized only with Mt-GFP. (Right) High-magnification images of single BY-2 cells bombarded with the vector shown at the right end. The images show overlaid images of green Mt-GFP and red MitoTracker. Yellow signals indicate areas where the green and red signals completely match. In the upper four images, all cells with and without wild-type ADL2b show normal mitochondrial morphology, with small spherical or peanut-shaped particles. In the bottom four images, all cells bombarded with mutant ADL2b (K56A or T77A) show fusion of mitochondria (fewer and longer mitochondria). [Bars = 10 μm.]

Figure 5

Tentative phylogenetic relationship showing parallel losses of MtFtsZ and the functional acquisition of a dynamin-like protein involved in mitochondrial division. This model assumes that (i) parallel losses of MtFtsZ occurred at least on the branch to higher plants and in the group of animals and fungi, and (ii) a functional acquisition of dynamin-like protein on mitochondrial division has occurred at the point before the divergence of these four groups. If this is correct, the common ancestor of green plants and red algae after the divergence of animals and fungi would have used both FtsZ and a dynamin-like protein in their mitochondrial division. It is possible that some presently living red algae have both proteins.