Mechanistic analysis of a dynamin effector - PubMed (original) (raw)

Mechanistic analysis of a dynamin effector

Laura L Lackner et al. Science. 2009.

Abstract

Dynamin-related proteins (DRPs) can generate forces to remodel membranes. In cells, DRPs require additional proteins [DRP-associated proteins (DAPs)] to conduct their functions. To dissect the mechanistic role of a DAP, we used the yeast mitochondrial division machine as a model, which requires the DRP Dnm1, and two other proteins, Mdv1 and Fis1. Mdv1 played a postmitochondrial targeting role in division by specifically interacting and coassembling with the guanosine triphosphate-bound form of Dnm1. This regulated interaction nucleated and promoted the self-assembly of Dnm1 into helical structures, which drive membrane scission. The nucleation of DRP assembly probably represents a general regulatory strategy for this family of filament-forming proteins, similar to F-actin regulation.

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Figures

Fig. 1

Fig. 1

Mdv1 preferentially interacts with the GTP-bound form of Dnm1. (A) Dnm1 and the indicated Dnm1 mutants at 0.4 μM were incubated with liposomes in the absence and presence of various nucleotides. The association of the protein with liposomes was assessed by its ability to float with liposomes after equilibrium sucrose gradient centrifugation. Equivalent amounts of the top (T) and bottom (B) fractions of the gradients were subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The percentage of protein found in the top fraction is shown as the mean and SEM; n = 3 independent experiments. (B) Dnm1 and Mdv1, each at 0.4 μM, were incubated with liposomes in the absence and presence of various nucleotides and subjected to liposome floatation and analysis as described in (A). Data are shown as the mean and SEM; n = 3 independent experiments. *P ≤ 0.05.

Fig. 2

Fig. 2

Mdv1 acts to nucleate Dnm1 self-assembly. (A) A plot of Dnm1 GTPase activity in the absence and presence of Mdv1 and OMC liposomes versus time after the initiation of self-assembly. The lag time as determined by linear regression analysis is shown in red and the steady-state region of the curve is shown in blue. Dnm1 and Mdv1 were present at 0.5 and 0.4 μM, respectively. OMC liposomes were present at 0.05 mg/ml, and GTP was present at 500 μM. (B) Quantification of lag time in the presence of various concentrations of Mdv1. Dnm1 was present at 1.5, 0.8, or 0.5 μM, as indicated, and GTP was present at 500 μM. Data are shown as the mean and SEM; n = 3 independent experiments. (C andD) Steady-state kinetics of Dnm1 in the absence and presence of Mdv1. Dnm1 and Mdv1 were present at 0.5 and 0.4 μM, respectively. A representative kinetic experiment is shown in (C). The table in (D) shows kinetic parameters determined as described in methods (32). _k_cat, turnover number; _K_0.5, substrate concentration where velocity is one-half maximal. Data are shown as the mean and SEM; _n_= 3 independent experiments. (E) Steady-state GTP hydrolysis activity of Dnm1 at various Dnm1 concentrations in the absence and presence of 0.4 μM Mdv1 and 0.05 mg/ml OMC liposomes, as indicated. GTP was present at 500 μM. Data are shown as the mean and SEM;n = 3 independent experiments.

Fig. 3

Fig. 3

Mdv1 stimulates Dnm1 self-assembly. (A) Velocity sedimentation of Dnm1 in the absence and presence of Mdv1. Dnm1 and Mdv1 were present at 0.2 μM. A representative Western blot is shown, and quantification data are shown as the mean and SEM; n = 3 independent experiments. (B and C) Analysis of GMPPCP-dependent Dnm1 self-assembly by negative-stain EM in the absence (left panels) and presence (right panels) of Mdv1. Different fields are shown in (B) and (C); the images in (C) were taken at a magnification 15,000 times higher than those in (B). Scale bars, 100 nm. (D) Negative-stain EM images of Dnm1-GMPPCP assembled on liposomes in the absence (left panel) and presence (right panel) of Mdv1. Scale bar, 100 nm. (E) Quantification of the distance from the inner edge of the lipid membrane to the outer edge of the assembled protein on Dnm1- and Dnm1-Mdv1 coated liposomes; _n_= 24 protein-coated liposomes. The mean and SEM are also shown.

Fig. 4

Fig. 4

Mdv1 oligomerizes. (A) Hydrodynamic parameters of Mdv1 were determined by sucrose gradient and gel filtration analyses and calculated as described in methods (32). (B) Analysis of Mdv1 oligomerization by chemical cross-linking. SDS-PAGE Coomassie-stained gel is shown of Mdv1 samples without (lane 1) and with (lane 2) a 5-M excess of cross-link reagent.

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