The Mechanoenzymatic Core of Dynamin-related Protein 1 Comprises the Minimal Machinery Required for Membrane Constriction

Abstract

Background: Drp1 oligomerization and activity is critical for mitochondrial fission. Results: GTP hydrolysis is required for Drp1 constriction of lipid bilayers. The variable domain of Drp1 regulates self-assembly and is not required for constriction of lipid bilayers. Conclusion:The core machinery of Drp1 is sufficient to mediate lipid assembly, constriction, and disassembly. Significance: Characterization of the mechanoenzymatic properties of Drp1 advances our understanding of mitochondrial fission.

Dynamin-related Protein 1 Oligomerization in Solution Impairs Functional Interactions with Membrane-anchored Mitochondrial Fission Factor

“…Protein Constructs and Mutagenesis-Drp1 Isoforms 1 and 3 (Drp1-1 and Drp1-3; UniProt IDs O00429-1 and O00429-4) and Drp1 Isoform 1 lacking residues 517-639 (⌬VD) were cloned into a pCal-n-EK vector with a human rhinovirus 3C protease (HR3CP) site as described previously (14). Mff lacking its transmembrane (TM) segment (Mff⌬TM; UniProt ID Q9GZY8-5, residues 1-218) and Mff lacking both its CC and its TM (Mff ⌬CC-TM; residues 1-186) were cloned into pET28a with a C-terminal His 6 affinity tag using NcoI and HindIII restriction sites introduced during PCR amplification.…”

“…1A): the GTPase domain, middle domain, variable domain (VD), and GTPase effector domain (GED). Hydrolysis of GTP triggers conformational changes in Drp1 oligomers that generate the mechanical force needed to promote mitochondrial membrane scission (14,15), and factors that inhibit Drp1 GTPase activity prevent mitochondrial division (8,16,17). The middle and GED domains promote Drp1 self-assembly, which is also critical for its role in facilitating mitochondrial fission (18,19).…”

“…The middle and GED domains promote Drp1 self-assembly, which is also critical for its role in facilitating mitochondrial fission (18,19). In vitro, the addition of negatively charged lipids increases Drp1 self-assembly to form larger helical assemblies that represent the contractile apparatus of mitochondrial fission (20), and these functional polymers exhibit stimulated GTPase activity (14,(21)(22)(23). The VD has recently been shown to act as a negative regulator of Drp1 self-assembly (14) with an inherent ability to interact with cardiolipin (CL) present in mitochondrial membranes (21,(23)(24)(25).…”

“…In vitro, the addition of negatively charged lipids increases Drp1 self-assembly to form larger helical assemblies that represent the contractile apparatus of mitochondrial fission (20), and these functional polymers exhibit stimulated GTPase activity (14,(21)(22)(23). The VD has recently been shown to act as a negative regulator of Drp1 self-assembly (14) with an inherent ability to interact with cardiolipin (CL) present in mitochondrial membranes (21,(23)(24)(25). Studies in yeast have shown that the VD is required for interactions with a mitochondrial adaptor protein (26), but the partner protein identified in that study is not conserved in higher eukaryotes, which suggests that the role of the VD may have evolved in higher organisms to accommodate different regulatory interactions in the cytosol and at the surface of mitochondria.…”

Structural Studies that Define Regulatory Interactions within the Mitochondrial Fission Machinery

“…To achieve this goal, our lab has examined Drp1 assemblies on various lipid templates using structural and functional studies [4][5][6]. There are similarities with other mechanochemical dynamins [7][8][9], but we continue to find novel attributes within the mammalian mitochondrial fission machinery.…”