The dynamin middle domain is critical for tetramerization and higher-order self-assembly

Abstract

The large multidomain GTPase dynamin self-assembles around the necks of deeply invaginated coated pits at the plasma membrane and catalyzes vesicle scission by mechanisms that are not yet completely understood. Although a structural role for the 'middle' domain in dynamin function has been suggested, it has not been experimentally established. Furthermore, it is not clear whether this putative function pertains to dynamin structure in the unassembled state or to its higher-order self-assembly or both. Here, we demonstrate that two mutations in this domain, R361S and R399A, disrupt the tetrameric structure of dynamin in the unassembled state and impair its ability to stably bind to and nucleate higher-order self-assembly on membranes. Consequently, these mutations also impair dynamin's assembly-dependent stimulated GTPase activity.

Figure 1

Dyn1-R361S and Dyn1-R399A are defective in self-assembly. (A) GTPase activities of 0.5 μM Dyn1-WT (○), Dyn-R361S (▪) or Dyn1-R399A (⧫) after pre-incubation with PI4,5P₂-containing liposomes (150 μM lipid) was measured at 37°C, as described under Materials and methods. The concentration of P_i released is plotted as a function of time. The average values of at least four independent experiments are shown with standard deviations, as indicated. (B) Self-assembly of dynamin (1 μM protein) on liposomes (300 μM lipid) with or without PI4,5P₂, or at low ionic strength (no salt), was examined by sedimentation followed by SDS–PAGE analysis of the supernatant (S) and pellet (P) fractions as described under Materials and methods; total protein (T). (C) Electron micrographs of Dyn1-WT, Dyn1-R361S and Dyn1-R399A (1 mg/ml protein) self-assembled on PI4,5P₂-containing liposomes (1 mg/ml lipid) were obtained as described under Materials and methods. Scale bar, 200 nm.

Figure 2

Dyn1-R361S and Dyn1-R399A are assembly-defective dimers. (A) Sedimentation coefficients (S) of Dyn1-WT (solid line), Dyn1-R361S (short dashes) and Dyn1-R399A (long dashes) at 5 μM each were obtained as described under Materials and methods and are plotted here as a function of the relative concentration of each species. (B) SEC elution profiles for Dyn1-WT (solid line), Dyn1-R361S (short dashes) and Dyn1-R399A (long dashes) run through a Superose 6 10/30 HR (Amersham Biosciences) column were obtained as described under Materials and methods. Protein absorbance at 280 nm (A₂₈₀) in milli-absorbance units (mAU) is plotted here as a function of elution volume (ml). Arrowhead points to the elution of dynamin microaggregates in the void volume.

Figure 3

Dyn1-R361S and Dyn1-R399A dimers exhibit impaired cooperativity in self-assembly. GTP hydrolysis rates of Dyn1-WT (○), Dyn-R361S (▪) or Dyn1-R399A (⧫) at low ionic strength (no salt) were determined at 37°C as described under Materials and methods, and are plotted as a function of dynamin concentration. The GTPase activities of Dyn1-R361S and Dyn1-R399A are linearly proportional to their concentration in contrast to Dyn1-WT, which exhibits sigmoidal behavior. The average values of at least three independent experiments are shown along with standard deviations.

Figure 4

Activation of dynamin basal GTPase is not due to nucleation. (A, B) The kinetics of basal GTP hydrolysis in Dyn1-WT (○) or Dyn1-R399A (⧫) at 2, 4 and 6 μM protein concentration was measured at 37°C using the GTP regenerating system as described under Materials and methods. The concentration of GTP hydrolyzed and released is plotted as a function of time. The average values of at least three independent experiments are shown. (C, D) Time-dependent emission intensity profiles for 0.1 μM Dyn1-WT^BODIPY (○) and Dyn1-R399A^BODIPY (⧫) before and upon addition (arrowhead) of either GTP (1 mM final) or liposomes (30 μM final) were obtained as described under Materials and methods. BODIPY was excited at 490 nm and emission was monitored at 510 nm. F₀ is the initial intensity of BODIPY at time ‘0' and F is its intensity at time ‘t'.