MiD51 and Mff Co-assemble in Cardiolipin-Enriched Membrane Microdomains to Cooperatively Regulate Drp1-Mediated Mitochondrial Fission

Abstract

Mitochondrial outer membrane (MOM)-anchored transmembrane protein adaptors, mitochondrial dynamics proteins of 49/51 kDa (MiD49/51) and mitochondrial fission factor (Mff), function to recruit the cytosolic mechanochemical GTPase, dynamin-related protein 1 (Drp1) toward mitochondrial fission. Whether these adaptors function independently or cooperatively in regulating Drp1 function remains controversial, as are their mechanisms in Drp1-catalyzed mitochondrial fission. MiD49/51 and Mff can independently recruit Drp1 to the mitochondrial surface, yet mitochondrial fission is impaired in each adaptor's absence suggesting that their functions are cooperative and non-redundant. Using reconstitution approaches in model membranes and multiple fluorescence spectroscopic and imaging techniques, we demonstrate that both MiD49/51 and Mff preferentially partition into cardiolipin (CL)-enriched membrane microdomains characteristic of MOM-MIM contact sites that regulate cristae remodeling and cytochrome c release during apoptotic mitochondrial fission. Surprisingly, MiD49 and MiD51 utilize their cytosolic nucleotidyltransferase domains to specifically bind CL. In the presence of CL, MiD49/51 co-assembles with Mff and Drp1 to constitute a ternary copolymer that is morphologically distinct from Drp1 assembled in the presence of either adaptor. We propose that MiD51 specifically recruits Drp1 dimers, which then associate with Mff to constitute a ternary polymeric complex that exhibits reduced Drp1 GTPase activity and helps retain the transition-state conformation of the Drp1 polymer on the membrane to effect membrane constriction. Thus, we propose MiD49/51 and Mff cooperatively regulate Drp1 function during apoptotic mitochondrial fission.