WD40 protein Mda1 is purified with Dnm1 and forms a dividing ring for mitochondria before Dnm1 in Cyanidioschyzon merolae

Abstract

Mitochondria are not produced de novo but are maintained by division. Mitochondrial division is a coordinated process of positioning and constriction of the division site and fission of double membranes, in which dynamin-related protein is believed to mediate outer membrane fission. Part of the mitochondrial division machinery was purified from M phase-arrested Cyanidioschyzon merolae cells through biochemical fractionation. The dynaminrelated protein Dnm1 was one of the two major proteins in the purified fraction and was accompanied by a newly identified protein CMR185C, named Mda1. Mda1 contained a predictable coiled-coil region and WD40 repeats, similarly to Mdv1 and Caf4 in yeasts. Immunofluorescence and immunoelectron microscopy showed that Mda1 localizes as a medial belt or ring on the mitochondrial outer surface throughout the division. The ring formation of Mda1 followed the plane of the ring of FtsZ, a protein that resides in the matrix. Dnm1 consistently colocalized with Mda1 only in the late stages of division. Mda1 protein was expressed through S to M phases and was phosphorylated specifically in M phase when Mda1 transformed from belt into foci and became colocalizing with Dnm1. Dephosphorylation of Mda1 in vitro increased its sedimentation coefficient, suggesting conformational changes of the macromolecule. Disassembly of the purified mitochondrial division machinery was performed by adding GTP to independently release Dnm1, suggesting that Mda1 forms a stable homo-oligomer by itself as a core structure of the mitochondrial division machinery.

Fission and Fusion of Plant Mitochondria, and Genome Maintenance

“…For studies of mitochondrial division, the cell cycle in C. merolae cells can be synchronized. The proteins that form mitochondrial division rings have been purified and identified (Yoshida et al, 2006;Nishida et al, 2007). C. merolae has two mitochondrial division proteins, an FtsZ homolog (CmFtsZ1) and a ZapA-like protein (ZED), that are similar to bacterial cell division proteins (Takahara et al, 2000;Yoshida et al, 2009).…”

The kinesin-like protein TOP promotes Aurora localisation and induces mitochondrial, chloroplast and nuclear division

“…Antibodies against Dynamin 1 (Dnm1), Dynamin 2 (Dnm2), Mda1, a-tubulin, CENH3 and mitochondrial porin were used as previously described Miyagishima et al, 2003;Nishida et al, 2005;Maruyama et al, 2007;Nishida et al, 2007;Fujiwara et al, 2009). Secondary antibodies used for immunofluorescence were, Alexa Fluor 488 or Alexa Fluor 555 goat anti-guinea pig, anti-mouse or anti-rabbit IgG, highly cross-adsorbed (Molecular Probes, Eugene, OR).…”

Single-membrane–bounded peroxisome division revealed by isolation of dynamin-based machinery

“…Dnm1 is one of only two dynamins (Dnm1 and Dnm2) encoded in the C. merolae genome (7,11). Dnm1 and Dnm2 are important components of the mitochondrion-dividing (MD) machinery (9,14,15) and plastiddividing (PD) machinery (16,17), respectively. Immunoblot analysis indicated that Dnm1 was concentrated in the dividing-peroxisome fraction with catalase ( Fig.…”

“…S6C). Fifth, the width of the filamentous ring in the POD machinery remained constant during peroxisome contraction, whereas those of the MD and PD machineries increased during contraction (8,15) (Figs. S6).…”