DataSheet1_The Molecular Assembly State of Drp1 Controls its Association With the Mitochondrial Recruitment Receptors Mff and MIEF1/2.docx

Drp1 is a central player in mitochondrial fission and is recruited to mitochondria by Mff and MIEFs (MIEF1 and MIEF2), but little is known about how its assembly state affects Drp1 mitochondrial recruitment and fission. Here, we used in vivo chemical crosslinking to explore the self-assembly state of Drp1 and how it regulates the association of Drp1 with MIEFs and Mff. We show that in intact mammalian cells Drp1 exists as a mixture of multiple self-assembly forms ranging from the minimal, probably tetrameric, self-assembly subunit to several higher order oligomers. Precluding mitochondria-bound Drp1 in Mff/MIEF1/2-deficient cells does not affect the oligomerization state of Drp1, while conversely forced recruitment of Drp1 to mitochondria by MIEFs or Mff facilitates Drp1 oligomerization. Mff preferentially binds to higher order oligomers of Drp1, whereas MIEFs bind to a wider-range of Drp1 assembly subunits, including both lower and higher oligomeric states. Mff only recruits active forms of Drp1, while MIEFs are less selective and recruit both active and inactive Drp1 as well as oligomerization- or GTPase-deficient Drp1 mutants to mitochondria. Moreover, all the fission-incompetent Drp1 mutants tested (except the monomeric mutant K668E) affect Drp1-driven mitochondrial dynamics via incorporation of the mutants into the native oligomers to form function-deficient Drp1 assemblies. We here confirm that MIEFs also serve as a platform facilitating the binding of Drp1 to Mff and loss of MIEFs severely impairs the interaction between Drp1 and Mff. Collectively, our findings suggest that Mff and MIEFs respond differently to the molecular assembly state of Drp1 and that the extent of Drp1 oligomerization regulates mitochondrial dynamics.

动力相关蛋白1（Dynamin-related protein 1, Drp1）是线粒体分裂的核心调控因子，可通过线粒体分裂因子（Mitochondrial fission factor, Mff）与MIEFs（MIEF1与MIEF2）被招募至线粒体，但目前对其组装状态如何影响Drp1的线粒体招募与分裂过程仍知之甚少。本研究采用体内化学交联技术，探究了Drp1的自组装状态及其对Drp1与MIEFs、Mff相互作用的调控作用。研究结果显示，在完整哺乳动物细胞内，Drp1以多种自组装形式共存，从最小的、推测为四聚体的自组装亚基直至多种高阶寡聚体均有分布。在缺失Mff/MIEF1/2的细胞中，阻断线粒体结合的Drp1并不会改变Drp1的寡聚化状态；反之，若通过MIEFs或Mff将Drp1强制招募至线粒体，则可显著促进Drp1的寡聚化进程。Mff优先结合Drp1的高阶寡聚体，而MIEFs的结合范围更广，可识别包括低阶、高阶寡聚体在内的多种Drp1组装亚基。Mff仅能招募具有活性的Drp1，相较而言MIEFs的选择性较弱，可将活性与非活性的Drp1，以及存在寡聚化缺陷或GTP酶（GTPase）活性缺陷的Drp1突变体均招募至线粒体。此外，本次检测的所有无法介导线粒体分裂的Drp1突变体（仅单体突变体K668E除外），均可通过掺入天然寡聚体形成功能缺陷的Drp1组装复合物，进而干扰Drp1调控的线粒体动态过程。本研究证实，MIEFs还可作为结合平台促进Drp1与Mff的相互作用，而MIEFs的缺失会严重削弱Drp1与Mff之间的相互作用。综上，本研究结果表明，Mff与MIEFs对Drp1的分子组装状态具有差异化响应，而Drp1的寡聚化程度可调控线粒体动态平衡。