Eukaryotes are defined by the presence of membrane-bounded subcellular organelles, which house specific biochemical reactions. In addition to performing distinct roles, various organelles can also function together in some intracellular metabolic pathways. In plants for example, peroxisomes, mitochondria, and chloroplasts act in concert in photorespiration (Peterhansel et al. 2010), lipid bodies, peroxisomes, mitochondria, and the cytosol together mediate the consecutive biochemical steps in fatty acid metabolism (Baker et al. 2006; Penfield et al. 2006), and jasmonic acid biosynthesis spans across chloroplasts and peroxisomes (Kazan and Manners 2008). Given the crucial roles of organelles in maintaining basic cellular functions, organelle population and distribution need to be tightly controlled. In plants, peroxisomes, mitochondria, and chloroplasts can divide by binary fission from pre-existing organelles, and one of the major division factors is the evolutionarily conserved dynamin-related protein, DRP (Yang et al. 2008; Kaur and Hu 2009; Logan 2010).
DRP belongs to the dynamin superfamily, which plays critical roles in diverse cellular processes such as vesicle scission, organelle fusion and fission, and cytokinesis (Praefcke and McMahon 2004). Classic dynamin proteins contain five conserved domains. The GTPase domain hydrolyzes guanosine triphosphate (GTP), the middle domain (MD) is responsible for self-interaction of dynamins through its coiled-coil region, the GTPase effector domain (GED) is involved in protein complex formation and activation of GTPase activity, the pleckstrin-homology domain (PHD) binds to negatively charged lipids, and the prolin-rich domain (PRD) provides the binding site for dynamin-binding proteins (Hinshaw 2000; Heymann and Hinshaw 2009). DRPs are defined by having at least the first three characteristic domains (Heymann and Hinshaw 2009); however, there are a few exceptions, in which the DRPs only contain the GTPase domain (Hong et al. 2003). Similar to dynamins, Dnm1p, a yeast DRP involved in peroxisomal and mitochondrial division, has GTPase activity and can self-assemble to form a spiral-like structure whose size matches the mitochondrial constriction sites (Ingerman et al. 2005). The assembled DRP polymers have been suggested to act as mechanochemical enzymes or signaling GTPases in a GTP hydrolysis-dependent manner (Praefcke and McMahon 2004). Recently, the three-dimensional (3D) structure of Dnm1p was solved using cryo-electron microscopy. In the presence of GTP, the Dnm1p spiral-like structure undergoes a large conformational change that presumably initiates constriction and fission of the mitochondrial membrane (Mears et al. 2011). The high-resolution crystal structure of human Dynamin1 (Drp1), a shared protein in mitochondrial and peroxisomal division, was also solved in two independent studies, which demonstrated that the higher-order formation of Drp1 is achieved through criss-cross assembly of the stalks (Faelber et al. 2011; Ford et al. 2011).
The Arabidopsis DRP family contains 16 members that can be divided into six subgroups (DRP1–6) based on their structural and sequence similarities and molecular activities (Hong et al. 2003). Among them, DRP1A and DRP1C have been suggested to function in clathrin-mediated membrane endocytosis (Collings et al. 2008; Konopka and Bednarek 2008; Fujimoto et al. 2010). DRP1C and DRP1E have also been implicated in mitochondrial morphogenesis (Jin et al. 2003). Like the DRP1s, DRP2A and DRP2B play a role in clathrin-mediated endocytosis (Bednarek and Backues 2010; Fujimoto et al. 2010; Taylor 2011). The DRP3 subfamily has two members, DRP3A and DRP3B, which are grouped in the same phylogenetic subclade as Drp1 and Dmn1p, peroxisomal and mitochondrial division factors from mammals and yeast (Saccharomyces cerevisiae) (Arimura and Tsutsumi 2002; Hong et al. 2003; Miyagishima et al. 2008). Consistent with this phylogenetic analysis, DRP3A and DRP3B are dual-localized and mediate the division of both peroxisomes and mitochondria (Arimura et al. 2004; Mano et al. 2004; Aung and Hu 2009; Fujimoto et al. 2009; Zhang and Hu 2009). Phosphorylation of DRP3A and DRP3B can also promote mitochondrial fission during mitosis (Wang et al. 2012). The DRP5 subfamily is composed of DRP5A and DRP5B. DRP5A is involved in cytokinesis (Miyagishima et al. 2008). DRP5B (ARC5), however, associates with chloroplasts and peroxisomes and mediates their division. Lack of a functional DRP5B (ARC5) leads to enlarged and dumbbell-shaped chloroplasts and highly aggregated peroxisomes that are impaired in fission (Gao et al. 2003; Zhang and Hu 2010).
DRP3A and DRP3B are partially redundant in the division of peroxisomes and mitochondria. However, despite having a small stature and mitochondria and peroxisomes that are grossly distorted morphologically, the drp3A drp3B double mutant is still fertile and contains both organelles in all cell types (Fujimoto et al. 2009; Zhang and Hu 2009). These data suggest that additional DRPs (such as DRP5B) or a DRP-independent machinery may function to maintain a low level of peroxisomal and mitochondrial division in the drp3 double mutant. In addition, although DRP3A, DRP3B, and DRP5B are all involved in peroxisomal division, it is unclear whether DRP5B's role is partially redundant to that of the DRP3s in this process, and whether DRP5B can form complexes with DRP3s in vivo. To address these questions, we generated a drp3A drp3B drp5B triple mutant, analyzed the impact of DRP5B on mitochondrial division, and tested the interaction among the three DRP proteins. Surprisingly, DRP5B affects mitochondrial division/morphogenesis in spite of lacking a stable physical association with this organelle. Furthermore, a DRP3-containing protein complex was detected in vivo in which DRP3A appears to be a major component. DRP5B does not directly interact with DRP3 in vitro, and is not a major component of the DRP3 protein complex. Our data have revealed the unexpected role of DRP5B in mitochondrial morphogenesis/division and DRP5B's DRP3-independent mode of action in organelle division.