Mitochondrial fission during mitophagy requires both inner and outer mitofissins

Mitophagy maintains mitochondrial homeostasis through selective degradation of damaged or excess mitochondria. Recently, we identified mitofissin/Atg44, a mitochondrial intermembrane space-resident fission factor, which directly acts on lipid membranes and drives mitochondrial fission required for mitophagy in yeast. However, it remains unclear whether mitofissin is sufficient for mitophagy-associated mitochondrial fission and whether other factors act from outside the mitochondria. Here, we identify a mitochondrial outer membrane-resident mitofissin-like microprotein required for mitophagy, and we name it mitofissin 2/Mfi2 based on the following results. Overexpression of a C-terminally truncated form of Mfi2 induces mitochondrial fragmentation and partially restores mitophagy in atg44 Δ cells. Mfi2 binds to lipid membranes and mediates membrane fission in vitro , demonstrating its intrinsic mitofissin activity. Genetic analyses reveal that Mfi2 and the dynamin-related protein Dnm1 independently facilitate mitochondrial fission during mitophagy. Thus, Atg44 and Mfi2, two mitofissins with distinct localizations, are required for mitophagy-associated mitochondrial fission.