ABSTRACT
The fusion of inner mitochondrial membranes requires dynamin-like GTPases, Mgm1 in yeast and OPA1 in mammals, but how they mediate membrane fusion is poorly understood. Here, we determined the crystal structure of Saccharomyces cerevisiae short Mgm1 (s-Mgm1) in complex with GDP. It revealed an N-terminal GTPase (G) domain followed by two helix bundles (HB1 and HB2) and a unique C-terminal lipid-interacting stalk (LIS). Dimers can form through antiparallel HB interactions. Head-to-tail trimers are built by intermolecular interactions between the G domain and HB2-LIS. Biochemical and in vivo analyses support the idea that the assembly interfaces observed here are native and critical for Mgm1 function. We also found that s-Mgm1 interacts with negatively charged lipids via both the G domain and LIS. Based on these observations, we propose that membrane targeting via the G domain and LIS facilitates the in cis assembly of Mgm1, potentially generating a highly curved membrane tip to allow inner membrane fusion.
Subject(s)
Crystallography, X-Ray , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/metabolism , Guanosine Diphosphate/chemistry , Mitochondria/enzymology , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , GTP-Binding Proteins/genetics , Guanosine Diphosphate/metabolism , Lipid Metabolism , Membrane Fusion , Mitochondrial Proteins/genetics , Models, Molecular , Mutation , Protein Conformation , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
The cytokinetic cleavage furrow is typically positioned symmetrically relative to the cortical cell boundaries, but it can also be asymmetric. The mechanisms that control furrow site specification have been intensively studied, but how polar cortex movements influence ultimate furrow position remains poorly understood. We measured the position of the apical and the basal cortex in asymmetrically dividing Drosophila neuroblasts and observed preferential displacement of the apical cortex that becomes the larger daughter cell during anaphase, effectively shifting the cleavage furrow toward the smaller daughter cell. Asymmetric cortical extension is correlated with the presence of cortical myosin II, which is polarized in neuroblasts. Loss of myosin II asymmetry by perturbing heterotrimeric G-protein signaling results in symmetric extension and equal-sized daughter cells. We propose a model in which contraction-driven asymmetric polar extension of the neuroblast cortex during anaphase contributes to asymmetric furrow position and daughter cell size.
Subject(s)
Asymmetric Cell Division , Drosophila melanogaster/cytology , Neural Stem Cells/cytology , Neurons/cytology , Animals , Brain/cytology , Brain/embryology , Cell Cycle Proteins/genetics , Cell Division , Cell Line , Cell Polarity , Cells, Cultured , Drosophila Proteins/metabolism , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , GTP-Binding Proteins/metabolism , Green Fluorescent Proteins , Microtubules/physiology , Microtubules/ultrastructure , Myosin Type II/metabolism , Neural Stem Cells/ultrastructure , Neurons/metabolism , Neurons/ultrastructure , Recombinant Fusion Proteins/metabolism , Signal Transduction , Spindle Apparatus/physiology , Spindle Apparatus/ultrastructureABSTRACT
Contractile force transduction by myosin II derives from its assembly into bipolar filaments. The coiled-coil tail domain of the myosin II heavy chain mediates filament assembly, although the mechanism is poorly understood. Tail domains contain an alternating electrostatic repeat, yet only a small region of the tail (termed the "assembly domain") is typically required for assembly. Using computational analysis, mutagenesis, and electron microscopy we discovered that the assembly domain does not function through self-interaction as previously thought. Rather, the assembly domain acts as a unique, positively charged interaction surface that can stably contact multiple complementary, negatively charged surfaces in the upstream tail domain. The relative affinities of the assembly domain to each complementary interaction surface sets the characteristic molecular staggers observed in myosin II filaments. Together these results explain the relationship between the charge repeat and assembly domain in stabilizing myosin bipolar filaments.
Subject(s)
Myosin Heavy Chains/chemistry , Myosin Type II/chemistry , Static Electricity , Animals , Computer Simulation , Drosophila , Microscopy, Electron , Mutagenesis , Myosin Type II/ultrastructure , Protein Conformation , Protein StabilityABSTRACT
Myosin II assembles into force-generating filaments that drive cytokinesis and the organization of the cell cortex. Regulation of myosin II activity can occur through modulation of filament assembly and by targeting to appropriate cellular sites. Here we show, using salt-dependent solubility and a novel fluorescence resonance energy transfer assay, that assembly of the Drosophila non-muscle myosin II heavy chain, zipper, is mediated by a 90-residue region (1849-1940) of the coiled-coil tail domain. This filament assembly domain, transiently expressed in Drosophila S2 cells, does not localize to the interphase cortex or the cytokinetic cleavage furrow, whereas a 500-residue region (1350-1865) that overlaps the NH(2) terminus of the assembly domain localizes to the interphase cortex but not the cytokinetic cleavage furrow. Targeting to these two sites appears to utilize distinct localization mechanisms as the assembly domain is required for cleavage furrow recruitment of a truncated coiled-coil tail region but not targeting to the interphase cortex. These results delineate the requirements for zipper filament assembly and indicate that the ability to form filaments is necessary for targeting to the cleavage furrow but not to the interphase cortex.
