Molecular mechanism of DRP1 assembly studied in vitro by cryo-electron microscopy.

Kaustuv Basu,Jin Chen,Blaise Bossy,Attila Sik,Ella Bossy-Wetzel,Isabelle Rouiller,Mihnea Bostina,Tristan Aumentado-Armstrong,Driss Lajoie,Roman I Koning,Hemachandra Reddy

doi:10.1371/journal.pone.0179397

Abstract

Mitochondria are dynamic organelles that continually adapt their morphology by fusion and fission events. An imbalance between fusion and fission has been linked to major neurodegenerative diseases, including Huntington’s, Alzheimer’s, and Parkinson’s diseases. A member of the Dynamin superfamily, dynamin-related protein 1 (DRP1), a dynamin-related GTPase, is required for mitochondrial membrane fission. Self-assembly of DRP1 into oligomers in a GTP-dependent manner likely drives the division process. We show here that DRP1 self-assembles in two ways: i) in the presence of the non-hydrolysable GTP analog GMP-PNP into spiral-like structures of ~36 nm diameter; and ii) in the presence of GTP into rings composed of 13−18 monomers. The most abundant rings were composed of 16 monomers and had an outer and inner ring diameter of ~30 nm and ~20 nm, respectively. Three-dimensional analysis was performed with rings containing 16 monomers. The single-particle cryo-electron microscopy map of the 16 monomer DRP1 rings suggests a side-by-side assembly of the monomer with the membrane in a parallel fashion. The inner ring diameter of 20 nm is insufficient to allow four membranes to exist as separate entities. Furthermore, we observed that mitochondria were tubulated upon incubation with DRP1 protein in vitro. The tubes had a diameter of ~ 30nm and were decorated with protein densities. These findings suggest DRP1 tubulates mitochondria, and that additional steps may be required for final mitochondrial fission.

Highlights

Mitochondria are dynamic organelles that frequently divide and fuse [1]
In the presence of GMP-PNP, DRP1 self-assembled into spiral-like structures with a diameter slightly larger than that of the rings (~36 nm) of variable length, reaching up to several microns (Fig 1C)
The most abundant rings were composed of 16 monomers having outer and inner ring diameters of ~30 nm and ~20 nm, respectively (Fig 1D)

Summary

Introduction

Mitochondria are dynamic organelles that frequently divide and fuse [1]. Mitochondrial division happens during cell division to distribute mitochondria to daughter cells and during apoptosis [2]. Members of the large GTPase dynamin superfamily, mechano-enzymes are responsible for mitochondrial fusion and fission [6]. Despite their functional diversity, all dynamin-related proteins (DRPs) are likely to undergo GTP-cycle-dependent conformational changes to drive self-assembly and disassembly [7,8,9,10,11,12]. Mitochondrial fission requires DRP1 in humans and its homolog dynamin 1 (Yeast Dnm1) in yeast [14,15,16]. Mutations resulting from deletion or loss of function of Yeast Dnm1/DRP-1 lead to extensive mitochondrial networks and are indicative of an increase in mitochondrial fusion [14,15,16,17,18,19]

Methods

Results

Conclusion