The Dynamin-Related Protein Mgm1p Assembles into Oligomers and Hydrolyzes GTP To Function in Mitochondrial Membrane Fusion

Abstract

Mitochondrial dynamics resulting from competing membrane fusion and fission reactions are required for normal cellular function in eukaryotes. Mgm1p, a dynamin-related protein, is a key component in yeast mitochondrial fusion and is evolutionarily conserved. Previous studies suggest that Mgm1p mediates mitochondrial inner membrane fusion in a manner similar to that of other dynamin proteins that use GTP hydrolysis and oligomerization to induce structural changes in lipid bilayers; however, a direct demonstration of these activities has yet to be presented. Here we show that purified Mgm1p forms low-order oligomers that are dependent on protein concentration, suggesting a dynamic and reversible interaction. We further demonstrate that Mgm1p has GTPase activity and kinetic properties consistent with a mechanoenzyme and with a role in inner membrane mitochondrial fusion. Mutations of key residues in conserved motifs of the GTPase domain show markedly reduced or diminished GTPase activity. A mutation in the GTPase effector domain, involved in assembly and assembly-stimulated GTP hydrolysis, has basal GTPase activity similar to that of wild-type Mgm1p but has a weaker propensity to form oligomers. Finally, our data indicate that Mgm1p interacts specifically with negatively charged phospholipids found in mitochondrial membranes, and point mutations in the predicted lipid-binding domain abrogate these interactions. These findings suggest the presence of a putative lipid-binding domain, providing insight into how this protein mediates inner membrane fusion. Together, these data indicate that Mgm1p mediates fusion through oligomerization, GTP hydrolysis, and lipid binding in a manner similar to those of other dynamin mechanoenzymes.

Prohibitins and the functional compartmentalization of mitochondrial membranes

“…Indeed, the processing of Mgm1 and the morphogenesis of cristae were identified as processes that require CL and PE within mitochondria (Osman et al, 2009;Sesaki et al, 2006). Consistently, Mgm1 binds anionic phospholipids including CL in vitro (Meglei and McQuibban, 2009). PHB complexes acting as membrane scaffolds might recruit proteases, or ensure a specific lipid environment that facilitates Mgm1 processing, and thereby prevent mitochondrial fragmentation under conditions of limited CL or PE.…”

Membrane Tethering and Nucleotide-dependent Conformational Changes Drive Mitochondrial Genome Maintenance (Mgm1) Protein-mediated Membrane Fusion

“…Protein Expression and Purification-s-Mgm1 was expressed and purified as described previously (20). In brief, cells were induced with 50 M isopropyl-1-thio-␤-D-galactopyranoside.…”

Dynamins at a glance

“…Purified yeast Mgm1 protein has been shown to assemble into low-order oligomers and displays GTPase activity. Although Mgm1 lacks a PH domain, it preferentially binds to negatively charged phospholipids that are typically found in mitochondrial membranes, which suggests that Mgm1 contains a lipid-binding motif (Meglei and McQuibban, 2009). Furthermore, the dynamins of the inner mitochondrial membrane, together with scaffolding proteins such as prohibitins, are thought to be involved in the maintenance of cristae morphology, possibly by assembling into higher-order structures (Merkwirth and Langer, 2008).…”