Abstract
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.
Highlights
Mitochondria are highly dynamic organelles that frequently change their shapes by shifting the balance between fission and fusion events in response to various cellular conditions
The results showed that neither Drp1 oligomerization-deficient nor GTPase-deficient mutants were recruited to mitochondria in the absence of endogenous MIEF1 and MIEF2
During the process of mitochondrial division, Drp1 is recruited from the cytosol to mitochondria, where it assembles into ring-like structures that consist of oligomeric Drp1 complexes which wrap around and constrict the mitochondrial tubule to drive mitochondrial fission (Yu et al, 2020)
Summary
Mitochondria are highly dynamic organelles that frequently change their shapes by shifting the balance between fission and fusion events in response to various cellular conditions. Mitochondrial fission is mediated by the highly conserved dynamin-related GTPase Drp. Drp primarily resides in the cytosol, but can be recruited to mitochondria through its mitochondrial receptors, Mff and MIEFs (MIEF1/MiD51 and MIEF2/MiD49), which are anchored in the mitochondrial outer membrane (Otera et al, 2010; Palmer et al, 2011; Zhao et al, 2011; Liu et al, 2013; Yu et al, 2017). Drp recruitment to mitochondria by Mff and MIEFs results in different outcomes: overexpression of Mff promotes fission leading to mitochondrial fragmentation, whereas overexpression of MIEF1 or MIEF2 inhibits fission resulting in mitochondrial elongation (Otera et al, 2010; Zhao et al, 2011; Liu et al, 2013; Loson et al, 2013)
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