Department of Oncology-Pathology, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden.
Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
J Biol Chem. 2019 Nov 15;294(46):17262-17277. doi: 10.1074/jbc.RA119.008202. Epub 2019 Sep 18.
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 (Drp1) 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 Drp1 to mitochondria and that elevated Mff or MIEF levels promote Drp1 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.
招募 GTPase 相关蛋白 1（Drp1）到线粒体是线粒体裂变所必需的关键步骤。可逆的 Drp1 磷酸化已被牵涉到这个过程的调控中，但 Drp1 在丝氨酸 637 上的磷酸化是否决定其亚细胞定位和裂变活性仍有待充分阐明。在这里，我们使用 HEK 293T 细胞和免疫荧光、免疫印迹、RNAi、亚细胞分级分离、共免疫沉淀测定和 CRISPR/Cas9 基因组编辑，表明磷酸化丝氨酸 637 上的 Drp1（Drp1）既存在于细胞质中，也存在于线粒体上。我们发现线粒体裂变因子（Mff）和线粒体延伸因子 1/2（MIEF1/2）的受体与 Drp1 相互作用并将其募集到线粒体，并且升高的 Mff 或 MIEF 水平促进 Drp1 在线粒体上的积累。我们还注意到，介导 Drp1 丝氨酸 637 磷酸化的蛋白激酶 A（PKA）部分存在于线粒体上，并与 MIEFs 和 Mff 相互作用。PKA 敲低不影响 Drp1-Mff 相互作用，但略微增强了 Drp1 与 MIEFs 的相互作用。在 Drp1 缺陷的 HEK 293T 细胞中，磷酸化模拟物 Drp1-S637D 和磷酸化缺陷物 Drp1-S637A 变体与野生型 Drp1 一样，定位于细胞质和线粒体，并挽救了 Drp1 缺陷引起的线粒体过度融合表型。然而，Drp1-S637D 诱导线粒体裂变的效率低于 Drp1-WT 和 Drp1-S637A。总之，Drp1 丝氨酸 637 磷酸化状态不是控制 Drp1 招募到线粒体的决定因素。
