Mgm1, the yeast ortholog of mammalian OPA1, is a key component in mitochondrial membrane fusion and is required for maintaining mitochondrial dynamics and morphology. We showed recently that the purified short isoform of Mgm1 (s-Mgm1) possesses GTPase activity, self-assembles into low order oligomers, and interacts specifically with negatively charged phospholipids (Meglei, Mitochondrial dynamics have been implicated in neurodegenerative diseases such as dominant optic atrophy and Parkinson disease (1, 2). Mitochondrial morphology is regulated by balanced membrane fusion and fission reactions that are orchestrated by members of the highly conserved dynaminrelated protein family (3). Dynamin-related proteins are large GTPases that can self-assemble and promote membrane remodeling (4, 5). We have shown previously that the dynaminrelated protein Mgm1 has GTPase activity, self-assembles into low order oligomers, and binds to negatively charged phospholipids (6). Mgm1 exists as two isoforms in the mitochondria; l-Mgm1 2 is anchored to the IM via a transmembrane domain, and s-Mgm1 is peripherally associated with the IM and also found in the intermembrane space. s-Mgm1 results from the regulated cleavage by the mitochondrial rhomboid protease (7,8). It was shown recently that both isoforms are essential but have distinct roles in mitochondrial membrane fusion whereby only s-Mgm1 requires its GTPase activity (9). It is proposed that l-Mgm1 serves as a receptor for s-Mgm1 to mediate fusion of opposing membranes upon GTP hydrolysis. Here, we provide molecular data indicating that lipid binding of s-Mgm1 is required for proper membrane fusion. Furthermore, structural analysis of s-Mgm1 assembled onto liposomes suggests a model whereby stacked trimers of s-Mgm1 on opposing membranes would facilitate fusion.G EXPERIMENTAL PROCEDURESExpression and Purification of s-Mgm1-WT s-Mgm1 and point mutants were purified in buffer containing 500 mM NaCl and 1 mM dithiothreitol as described previously (6). For all subsequent in vitro assays presented here, purified WT s-Mgm1 and point mutants were diluted into buffers containing 170 mM NaCl and 1 mM dithiothreitol immediately prior to the assay.Liposome Preparation-A 10 mg/ml chloroform solution of lipid (Avanti Polar Lipids) was dried by rotary evaporation and vacuum pump, yielding a thin lipid film. The lipid suspension in physiological salt buffer was extruded 15 times through a 1-m polycarbonate Nucleopore TM track-etched membrane (Whatman) to generate unilamellar vesicles.Enzyme-linked Immunosorbent Assay-IM liposomes were prepared with the corresponding physiological concentrations: cardiolipin, 16%; phosphatidylethanolamine, 24%; phosphatidic acid, 2%; phosphatidylserine, 4%; phosphatidylcholine, 38%; and phosphatidylinositol, 16%. 2 g of total lipid was added to each well of a 96-well plate and allowed to coat overnight. Following 5% bovine serum albumin block, Mgm1 proteins were added at the concentrations indicated. Binding was detected using primary antibody directed against ...

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.

We previously reported the construction of metagenomic libraries in the IncP cosmid vector pRK7813, enabling heterologous expression of these broad-host-range libraries in multiple bacterial hosts. Expressing these libraries in Sinorhizobium meliloti, we have successfully complemented associated phenotypes of polyhydroxyalkanoate synthesis mutants. DNA sequence analysis of three clones indicates that the complementing genes are homologous to, but substantially different from, known polyhydroxyalkanaote synthase-encoding genes. Thus we have demonstrated the ability to isolate diverse genes for polyhydroxyalkanaote synthesis by functional complementation of defined mutants. Such genes might be of use in the engineering of more efficient systems for the industrial production of bioplastics. The use of functional complementation will also provide a vehicle to probe the genetics of polyhydroxyalkanaote metabolism and its relation to carbon availability in complex microbial assemblages.