Dnm1 forms spirals that are structurally tailored to fit mitochondria

Abstract

Dynamin-related proteins (DRPs) are large self-assembling GTPases whose common function is to regulate membrane dynamics in a variety of cellular processes. Dnm1, which is a yeast DRP (Drp1/Dlp1 in humans), is required for mitochondrial division, but its mechanism is unknown. We provide evidence that Dnm1 likely functions through self-assembly to drive the membrane constriction event that is associated with mitochondrial division. Two regulatory features of Dnm1 self-assembly were also identified. Dnm1 self-assembly proceeded through a rate-limiting nucleation step, and nucleotide hydrolysis by assembled Dnm1 structures was highly cooperative with respect to GTP. Dnm1 formed extended spirals, which possessed diameters greater than those of dynamin-1 spirals but whose sizes, remarkably, were equal to those of mitochondrial constriction sites in vivo. These data suggest that Dnm1 has evolved to form structures that fit the dimensions of mitochondria.

Figure 1.

Kinetics and velocity sedimentation of Dnm1. (A) Steady-state kinetics of Dnm1 GTPase activity under assembly conditions. 0.06 mg/ml Dnm1 was assayed for GTPase activity at 150 mM NaCl. (B) Velocity sedimentation of Dnm1 at low and high ionic strength. T, total; S, supernatant; P, pellet.

Figure 2.

High ionic strength antagonizes Dnm1 self-assembly and reveals GTP-dependent cooperation. (A) Steady-state kinetics of Dnm1 at high ionic strength. 0.06 mg/ml Dnm1 was assayed for GTPase activity at 500 mM NaCl. (B) Gel filtration analysis of Dnm1 using a Superose 6 column at 750 mM NaCl. (C) Sucrose gradient sedimentation of Dnm1 (from which the blot was created). Fractions collected from a 5–20% sucrose gradient were analyzed by Western blot analysis using anti-Dnm1 antibodies. Native high molecular mass markers were used to create a standard curve. Sedimentation coefficients of the protein standards were plotted versus the mean sucrose gradient fraction number in which the protein standard appeared. Molecular mass standards with sedimentation coefficients in parentheses that were distributed in sucrose gradient fractions are listed as follows (top, fraction 1; bottom, fraction 10): BSA (4.3 S), fraction 2.3; lactate dehydrogenase (7.4 S), fraction 3.3; catalase (11.3 S), fraction 4.6; and thyroglobulin (19.4 S), fraction 6.9. P, pellet.

Figure 3.

Nucleation-dependent assembly of Dnm1. (A) Time dependence of Dnm1 GTPase activity at low ionic strength. GTPase assay was initiated by the dilution of Dnm1at high ionic strength into low ionic strength GTPase assay buffer. (B) Dnm1 concentration dependence of lag time. Indicated concentrations of Dnm1 were assayed at low ionic strength. Only steady-state regions of plots of GTPase activity versus time are shown to demonstrate a decrease in lag time with increasing Dnm1 concentration.

Figure 4.

Dnm1G385D is an assembly-deficient dimer. (A) Steady-state kinetics of 0.08 mg/ml Dnm1G385D at 150 mM NaCl. (B) Gel filtration analysis of Dnm1G385D using a Superose 6 column at 500 mM NaCl. (C) Sucrose gradient sedimentation of Dnm1G385D (from which the blot was created). Fractions collected from a 10–30% sucrose gradient were analyzed as described in Fig. 2. Molecular mass standards with sedimentation coefficients stated in parentheses that were distributed in sucrose gradient fractions are listed as follows (top, fraction 1; bottom, fraction 10): BSA (4.3 S), fraction 1.6; lactate dehydrogenase (7.4 S), fraction 2.5; catalase (11.3 S), fraction 3.5; and thyroglobulin (19.4 S), fraction 5.1. (D) Time dependence of Dnm1G385D GTPase activity under experimental conditions that were similar to those of wild-type Dnm1.

Figure 5.

Dnm1 self-assembles into spirals that are structurally tailored to fit mitochondria. (A–C) Electron micrographs of negatively stained Dnm1 structures. (A) Dnm1 forms curved filaments in the absence of nucleotides. (B) Dnm1 self-assembles into large spirals in the presence of GMP-PCP. (Inset) Dynamin-1 spirals formed in the presence of GDP/BeF_x. (C) Dnm1/GMP-PCP spirals undergo a conformational change upon the addition of GTP. (D) Conventional EM analysis of mitochondrial constriction sites in thin sections of yeast cells. Arrowheads indicate electron-dense structures that are found in association with mitochondrial constriction sites. M, matrices of the mitochondria. (E) Dnm1 assembly in the presence of liposomes. Bars, 100 nm.