Mitochondrial dynamics is a fundamental cellular process and recruitment of Drp1 to mitochondria is an essential step in mitochondrial fission. Mff and MIEF1/2 (MiD51/49) serve as key receptors for recruitment of Drp1 to mitochondria in mammals. However, if and how these receptors work together in mitochondrial fission is poorly understood. Here we show that MIEFs interact with both Drp1 and Mff on the mitochondrial surface and serve as adaptors linking Drp1 and Mff together in a trimeric Drp1-MIEF-Mff complex. Thus, MIEFs can regulate the interaction between Drp1 and Mff, and also Mffinduced Drp1 accumulation on mitochondria. It is shown that loss of endogenous MIEFs severely impairs these processes. Additionally, in cells depleted of endogenous MIEF1/2, high levels of exogenous MIEFs sequester Drp1 on the mitochondrial surface, resulting in mitochondrial elongation, whereas low-tomoderate levels of MIEFs promote mitochondrial fission, leading to mitochondrial fragmentation. In sum, the data suggest that MIEFs and Mff work coordinately in Drp1-mediated mitochondrial fission and that the level of MIEF1/2 relative to Mff sets the balance between mitochondrial fission and fusion.Cells need to regulate the morphology of mitochondria in response to various physiological challenges and the dynamin-related GTPase Drp1 has emerged as a central regulator in mitochondrial fission. Drp1 is primarily distributed in the cytoplasm, but shuttles between the cytoplasm and mitochondria 1, 2 . Drp1 recruitment from the cytoplasm to the mitochondrial outer membrane (MOM) is an essential step in mitochondrial fission [3][4][5] . At the MOM, Drp1 is assembled into helical structures that wrap around the mitochondria to induce mitochondrial fission via its GTPase activity 1,5,6 . Several proteins located at the MOM, including Fis1, Mff and MIEFs (MIEF1 and MIEF2, also known as MiD51/MiD49) have been identified as receptors for the recruitment of Drp1 to mitochondria in mammals. While Fis1 was the first proposed Drp1 receptor at the MOM 7,8 , several recent studies suggest that Fis1 plays only a minor role in Drp1 recruitment [9][10][11] . Mff and MIEFs have been identified as alternative receptors for Drp1 9, 12, 13 . Despite they both function independently as receptors to bind and recruit cytosolic Drp1 to the mitochondrial surface, Mff and MIEFs have opposing effects on mitochondrial morphology following exogenous expression: overexpression of Mff results in excessive mitochondrial fragmentation 9, 14 , whereas overexpression of MIEF1 or MIEF2 leads to mitochondrial elongation most likely by inhibiting fission [11][12][13] . Thus, it is believed that Mff is the primary receptor for Drp1 to facilitate mitochondrial fission 9,11,14,15 , whereas MIEFs recruit but presumably suppress Drp1's function by sequestering the protein in an inactive state on the mitochondrial surface 11,13,16 . Although Mff, MIEF1 and MIEF2 as well as hFis1 are known to be simultaneously expressed in cells 17,18 , it is unclear whether and how these receptor...

Recruitment of the GTPase dynamin-related protein 1 (Drp1) to mitochondria is a central step required for mitochondrial fission. Reversible Drp1 phosphorylation has been implicated in the regulation of this process, but whether Drp1 phosphorylation at Ser-637 determines its subcellular localization and fission activity remains to be fully elucidated. Here, using HEK 293T cells and immunofluorescence, immunoblotting, RNAi, subcellular fractionation, co-immunoprecipitation assays, and CRISPR/Cas9 genome editing, we show that Drp1 phosphorylated at Ser-637 (Drp1pS637) resides both in the cytosol and on mitochondria. We found that the receptors mitochondrial fission factor (Mff) and mitochondrial elongation factor 1/2 (MIEF1/2) interact with and recruit Drp1pS637 to mitochondria and that elevated Mff or MIEF levels promote Drp1pS637 accumulation on mitochondria. We also noted that protein kinase A (PKA), which mediates phosphorylation of Drp1 on Ser-637, is partially present on mitochondria and interacts with both MIEFs and Mff. PKA knockdown did not affect the Drp1-Mff interaction, but slightly enhanced the interaction between Drp1 and MIEFs. In Drp1-deficient HEK 293T cells, both phosphomimetic Drp1-S637D and phospho-deficient Drp1-S637A variants, like wild-type Drp1, located to the cytosol and to mitochondria and rescued a Drp1 deficiency-induced mitochondrial hyperfusion phenotype. However, Drp1-S637D was less efficient than Drp1-WT and Drp1-S637A in inducing mitochondrial fission. In conclusion, the Ser-637 phosphorylation status in Drp1 is not a determinant that controls Drp1 recruitment to mitochondria.