Abstract
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.
Highlights
Recruitment of the GTPase dynamin-related protein 1 (Drp1) to mitochondria is a central step required for mitochondrial fission
Several studies have suggested that Drp1 phosphorylation at residue Ser-637 by protein kinase A (PKA) inhibits mitochondrial fission by decreasing the intramolecular interactions that normally drive GTPase activity and by preventing translocation of Drp1 to mitochondria, whereas dephosphorylation at Ser-637 increases mitochondrial recruitment of Drp1 and promotes mitochondrial fission (44 – 46, 54)
It was not established in those studies whether the phosphorylation status at Drp1–Ser-637 is a determinant directly controlling the mitochondrial recruitment of Drp1
Summary
Mitochondrial fission factor; MIEF, mitochondrial elongation factor; PCC, Pearson’s correlation coefficient; pAb, polyclonal antibody; co-IP, co-immunoprecipitation. Phosphorylation of Drp is the most studied posttranslational modification regulating mitochondrial fission, and phosphorylation occurs at two serine residues: Ser-616 and Ser-637 (in human Drp isoform 1) [33, 39, 40]. Reversible phosphorylation of Drp may play a critical role in regulating its subcellular localization and fission activity, but many aspects of Drp phosphorylation are still not fully understood. We address the role of Drp phosphorylation at Ser-637 (Drp1pS637) for subcellular localization and ability to regulate mitochondrial fission. We show that Drp1pS637 distributes to both the cytosol and mitochondria and does not play a major role in controlling mitochondrial fission
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