A dimeric equilibrium intermediate nucleates Drp1 reassembly on mitochondrial membranes for fission

Abstract

The GTPase dynamin-related protein 1 (Drp1) catalyzes mitochondrial division, but the mechanisms remain poorly understood. Much of what is attributed to Drp1's mechanism of action in mitochondrial membrane fission parallels that of prototypical dynamin in endocytic vesicle scission. Unlike the case for dynamin, however, no lipid target for Drp1 activation at the mitochondria has been identified. In addition, the oligomerization properties of Drp1 have not been well established. We show that the mitochondria-specific lipid cardiolipin is a potent stimulator of Drp1 GTPase activity, as well as of membrane tubulation. We establish further that under physiological conditions, Drp1 coexists as two morphologically distinct polymeric species, one nucleotide bound in solution and the other membrane associated, which equilibrate via a dimeric assembly intermediate. With two mutations, C300A and C505A, that shift Drp1 polymerization equilibria in opposite directions, we demonstrate that dimers, and not multimers, potentiate the reassembly and reorganization of Drp1 for mitochondrial membrane remodeling both in vitro and in vivo.

FIGURE 1:

Cardiolipin potently stimulates Drp1 GTPase activity. (A) Domain architecture of Drp1 and a color-coded representation of a monomer in the dimer crystal structure (Protein Data Bank ID 4BEJ). Residues mutated in this study are indicated and represented in space-fill. BSE, bundle signaling element; G, GTPase domain; GED; GTPase effector domain. (B) GTPase activity of Drp1 (0.5 μM) in the absence and presence of CL-containing liposomes (150 μM total lipid) plotted as concentration of P_i released over time. The average turnover number (k_cat) ± SD is indicated. (C) Specific activity of Drp1 on CL-containing liposomes (150 μM total lipid) as a function of protein concentration. Average ± SD for n ≥ 3. (D) Specific activity of Drp1 (0.5 μM) on CL-containing liposomes (150 μM total lipid) as a function of mole percent CL content. Average ± SD for n ≥ 3.

FIGURE 2:

Drp1 efficiently remodels CL-containing membranes. (A) Binding of BODIPY-labeled Drp1 (green channel) to PC/CL-containing, rhodamine-labeled GUVs (red channel). PC-only control GUVs do not bind Drp1. The final Drp1 concentration was 0.5 μM. Scale bar, 5 μm. (B) Time-lapse series of membrane tubulation by BODIPY-Fl–labeled Drp1 (0.5 μM protein final) on rhodamine-labeled PC/PE/CL GUVs. Scale bar, 5 μm. (C) Representative low- magnification (scale bar, 500 nm) and high-magnification (scale bar, 100 nm) EM images of Drp1-decorated membrane tubules.

FIGURE 3:

Morphologically distinct polymers of Drp1. (A) Tightly wound helical polymers of Drp1 formed in the presence of GMP-PCP in solution. (B) Coexisting solution-suspended (slender arrows) and membrane-associated (block arrows) helical polymers of Drp1 found in the presence of GMP-PCP and CL-containing liposomes. Representative EM images. Scale bar, 100 nm.

FIGURE 4:

Drp1 polymerization equilibria and mutants. (A) SEC-MALS analyses of Drp1 WT and mutants. Middle, determined molar mass profiles (black dots) for discernible peak regions of the broad Drp1 elution profile. Differential refractive indices (dRI) are normalized and plotted against elution volume (ml). (B) SEC elution profile shifts for Drp1 WT and mutants as a function of loaded protein concentration. Solid, dotted, and dashed line profiles represent loaded protein concentrations of 10, 5, and 2.5 μM, respectively. Normalized ultraviolet light absorbance measured at 280 nm is plotted against elution volume (ml). Arrows point to corresponding peak positions for Drp1 dimers, tetramers, and hexamers. (C) EM images of Drp1 WT and mutant helical polymers formed in the presence of GMP-PCP in solution. Scale bar, 200 nm. Middle, inset, magnified image of a Drp1 C300A helical polymer. Scale bar (white), 50 nm. (D) Percentage Drp1 sedimented by high-speed centrifugation in the absence or presence of GMP-PCP or GTP for WT and mutants. Averages ± SD for n ≥ 3.

FIGURE 5:

Drp1 dimers potentiate membrane remodeling. (A) Left, FRET-sensitized, time-dependent increase in Dansyl emission intensity upon Trp excitation plotted as F/F₀, where F₀ is the background Dansyl emission intensity before Drp1 addition, and F is Dansyl emission intensity at time t after Drp1 addition. Representative traces. Right, percentage Drp1 sedimented by high-speed centrifugation after incubation with CL-containing liposomes. Averages ± SD for n ≥ 3. (B) Representative confocal fluorescence images of membrane tubules extracted by BODIPY-Fl–labeled Drp1 and mutants (green) from rhodamine–labeled PC/CL GUVs (red). Scale bar, 5 μm. The relative efficiency of GUV tubulation is quantified and indicated as percentage GUVs tubulated. More than 25 GUVs were scored for each Drp1 species from three independent experiments. Numbers in parentheses represent those from PC/PE/CL GUVs. (C) Specific activity of Drp1 and mutants on CL-containing liposomes (150 μM total lipid) as a function of protein concentration. Averages ± SD for n ≥ 3. (D) Model for Drp1 polymerization equilibria and morphology. Drp1 C505A exists predominantly as “dimers” in the cytosol, whereas Drp1 C300A is stabilized as “supra-oligomers.”

FIGURE 6:

Drp1 dimers rescue mitochondrial fission. (A) Confocal fluorescence imaging of mitochondrial morphology in Drp1 KO MEFs expressing either Myc-tagged Drp1 WT or mutants. Block arrows point to either fused or fragmented mitochondria. Inset (white box) shows a magnified view of boxed region (yellow) within each representative cell image. (B) Quantification of mitochondrial fission in Drp1 KO MEFs expressing either Myc-tagged Drp1 WT or mutants. More than 100 Myc-positive cells from three independent experiments were scored for each sample. Data are represented as mean ± SEM. (C) Representative Western blot showing expression levels of Myc-tagged Drp1 WT and mutants in transfected Drp1 KO MEFs. Untransfected cell lysate (– –) was used as negative control, with actin serving as loading control for total protein.