Phospholipid Association Is Essential for Dynamin-related Protein Mgm1 to Function in Mitochondrial Membrane Fusion

Abstract

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 ...

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

“…EM images as well as two-dimensional crystallization analysis of the lattices by Fourier transformation and image processing showed a hexameric (flower-like) (26) three-fold symmetry lattice (Figs. 1F and 3, A and B, left panels), consistent with two previous studies (17,18). A structural transition in the crystalline array to a square lattice is observed by the addition of 1 mM GTP and/or its nonhydrolyzable analog GMPPCP (Fig.…”

“…Tethering-Using liposomes reflecting IM lipid composition, we have previously demonstrated by negative stain electron microscopy that s-Mgm1 assembled onto liposomes and promoted liposome aggregation (17). We have extended and refined our characterization of s-Mgm1-membrane associations.…”

“…Liposome Preparation-Liposomes were prepared from 1,2-dioleoyl-sn-glycero-3-phospho-L-serine and inner mitochondrial (IM) phospholipid composition as follows: 38% 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 24% 1,2-dimyristoylsn-glycero-3-phosphoethanolamine, 16% L-␣-phosphatidylinositol, 16% 1Ј,3Ј-bis[1,2-dimyristoyl-sn-glycero-3-phospho]-sn-glycerol, 4% 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine, and 2% 1,2-dimyristoyl-sn-glycero-3-phosphate, as described previously (17).…”

“…The mechanism of action of the protein has yet to be determined, but requires two isoforms, an inner membrane-bound l-Mgm1 and an intermembrane space s-Mgm1; both isoforms contain a lipid-binding domain that is required for their in vivo function (17,18). Recent studies showed that a functional GTPase domain is only required for s-Mgm1 function, and with their differential localizations along the mitochondrial inner membrane, both isoforms are proposed to have distinct roles (19).…”

Proteome-wide detection of phospholipid–protein interactions in mitochondria by photocrosslinking and click chemistry

“…More recently, CL has been implicated in the interaction of the major ADP/ATP carrier protein Aac2p with other membrane protein complexes in the mitochondrial inner membrane, 22 and in mitochondrial inner membrane fusion through its interaction with the dynamin-related protein Mgm1. 23,24 Moreover, CL has been implicated in the stability of a supercomplex of the prohibitin and the m-AAA protease complexes. 25 The other major structural phospholipids PC, PE and PI have also been identified in the crystal structures of respiratory complexes III and IV, 17,18 suggesting that they may be important for the structure and activity of these proteins.…”