ABSTRACT
Mitochondria are double-membrane-bound organelles that constantly change shape through membrane fusion and fission. Outer mitochondrial membrane fusion is controlled by Mitofusin, whose molecular architecture consists of an N-terminal GTPase domain, a first heptad repeat domain (HR1), two transmembrane domains, and a second heptad repeat domain (HR2). The mode of action of Mitofusin and the specific roles played by each of these functional domains in mitochondrial fusion are not fully understood. Here, using a combination of in situ and in vitro fusion assays, we show that HR1 induces membrane fusion and possesses a conserved amphipathic helix that folds upon interaction with the lipid bilayer surface. Our results strongly suggest that HR1 facilitates membrane fusion by destabilizing the lipid bilayer structure, notably in membrane regions presenting lipid packing defects. This mechanism for fusion is thus distinct from that described for the heptad repeat domains of SNARE and viral proteins, which assemble as membrane-bridging complexes, triggering close membrane apposition and fusion, and is more closely related to that of the C-terminal amphipathic tail of the Atlastin protein.
Subject(s)
GTP Phosphohydrolases/physiology , Membrane Fusion , Mitochondria/physiology , Mitochondrial Dynamics , Mitochondrial Membrane Transport Proteins/physiology , Mitochondrial Proteins/physiology , Animals , Cells, Cultured , GTP Phosphohydrolases/chemistry , GTP Phosphohydrolases/genetics , Lipid Bilayers/metabolism , Mice , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/genetics , Protein DomainsABSTRACT
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins constitute the core membrane fusion machinery of intracellular transport and intercellular communication. A little more than ten years ago, it was proposed that the long N-terminal domain of a subset of SNAREs, henceforth called the longin domain, could be a crucial regulator with multiple functions in membrane trafficking. Structural, biochemical and cell biology studies have now produced a large set of data that support this hypothesis and indicate a role for the longin domain in regulating the sorting and activity of SNAREs. Here, we review the first decade of structure-function data on the three prototypical longin SNAREs: Ykt6, VAMP7 and Sec22b. We will, in particular, highlight the conserved molecular mechanisms that allow longin domains to fold back onto the fusion-inducing SNARE coiled-coil domain, thereby inhibiting membrane fusion, and describe the interactions of longin SNAREs with proteins that regulate their intracellular sorting. This dual function of the longin domain in regulating both the membrane localization and membrane fusion activity of SNAREs points to its role as a key regulatory module of intracellular trafficking.
Subject(s)
SNARE Proteins , Animals , Biological Transport, Active/physiology , Humans , Protein Structure, Tertiary , SNARE Proteins/chemistry , SNARE Proteins/genetics , SNARE Proteins/metabolism , Structure-Activity RelationshipABSTRACT
The conditional use of actin during clathrin-mediated endocytosis in mammalian cells suggests that the cell controls whether and how actin is used. Using a combination of biochemical reconstitution and mammalian cell culture, we elucidate a mechanism by which the coincidence of PI(4,5)P2 and PI(3)P in a curved vesicle triggers actin polymerization. At clathrin-coated pits, PI(3)P is produced by the INPP4A hydrolysis of PI(3,4)P2, and this is necessary for actin-driven endocytosis. Both Cdc42â guanosine triphosphate and SNX9 activate N-WASP-WIP- and Arp2/3-mediated actin nucleation. Membrane curvature, PI(4,5)P2, and PI(3)P signals are needed for SNX9 assembly via its PX-BAR domain, whereas signaling through Cdc42 is activated by PI(4,5)P2 alone. INPP4A activity is stimulated by high membrane curvature and synergizes with SNX9 BAR domain binding in a process we call curvature cascade amplification. We show that the SNX9-driven actin comets that arise on human disease-associated oculocerebrorenal syndrome of Lowe (OCRL) deficiencies are reduced by inhibiting PI(3)P production, suggesting PI(3)P kinase inhibitors as a therapeutic strategy in Lowe syndrome.
Subject(s)
Actins/metabolism , Clathrin-Coated Vesicles/metabolism , Clathrin/metabolism , Coated Pits, Cell-Membrane/metabolism , Endocytosis , Phosphatidylinositols/metabolism , Actin-Related Protein 2-3 Complex/genetics , Actin-Related Protein 2-3 Complex/metabolism , Animals , CRISPR-Cas Systems , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , HeLa Cells , Humans , Hydrolysis , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Oculocerebrorenal Syndrome/genetics , Oculocerebrorenal Syndrome/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphatidylinositol Phosphates/metabolism , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/metabolism , Protein Multimerization , RNA Interference , Retinal Pigment Epithelium/metabolism , Signal Transduction , Sorting Nexins/genetics , Sorting Nexins/metabolism , Time Factors , Transfection , Wiskott-Aldrich Syndrome Protein, Neuronal/genetics , Wiskott-Aldrich Syndrome Protein, Neuronal/metabolism , Xenopus laevis , cdc42 GTP-Binding Protein/genetics , cdc42 GTP-Binding Protein/metabolismABSTRACT
Curving biological membranes establishes the complex architecture of the cell and mediates membrane traffic to control flux through subcellular compartments. Common molecular mechanisms for bending membranes are evident in different cell biological contexts across eukaryotic phyla. These mechanisms can be intrinsic to the membrane bilayer (either the lipid or protein components) or can be brought about by extrinsic factors, including the cytoskeleton. Here, we review examples of membrane curvature generation in animals, fungi, and plants. We showcase the molecular mechanisms involved and how they collaborate and go on to highlight contexts of curvature that are exciting areas of future research. Lessons from how membranes are bent in yeast and mammals give hints as to the molecular mechanisms we expect to see used by plants and protists.
Subject(s)
Cell Membrane/metabolism , Animals , Biomechanical Phenomena , Cell Shape , Endosomes/metabolism , Humans , Models, Biological , Secretory PathwayABSTRACT
Development of the nervous system requires extensive axonal and dendritic growth during which neurons massively increase their surface area. Here we report that the endoplasmic reticulum (ER)-resident SNARE Sec22b has a conserved non-fusogenic function in plasma membrane expansion. Sec22b is closely apposed to the plasma membrane SNARE syntaxin1. Sec22b forms a trans-SNARE complex with syntaxin1 that does not include SNAP23/25/29, and does not mediate fusion. Insertion of a long rigid linker between the SNARE and transmembrane domains of Sec22b extends the distance between the ER and plasma membrane, and impairs neurite growth but not the secretion of VSV-G. In yeast, Sec22 interacts with lipid transfer proteins, and inhibition of Sec22 leads to defects in lipid metabolism at contact sites between the ER and plasma membrane. These results suggest that close apposition of the ER and plasma membrane mediated by Sec22 and plasma membrane syntaxins generates a non-fusogenic SNARE bridge contributing to plasma membrane expansion, probably through non-vesicular lipid transfer.