Membrane fusion is vitally important for many aspects of eukaryotic cell biology including vesicular traffic within the secretory pathway as well as the biogenesis and maintenance of the entire endomembrane system. Membrane fusion also provides cytoplasmic organelles like mitochondria and the endoplasmic reticulum the ability to change shape and size to perform their required function. This proposal examines the role of the GTPase atlastin in homotypic ER fusion. Atlastin (SPG3A) is a member of a larger family of genes that are responsible for a group of inherited neurological disorders called Hereditary Spastic Paraplegias (HSP). Mutations in atlastin-1 account for ~10% of autosomal dominant forms of HSP. A fundamental understanding of atlastin's role in generating and maintaining ER function by homotypic ER fusion will significantly inform the mechanistic basis of ER-associated pathologies such as the neuronal degeneration found in HSP. Atlastin utilizes the chemical energy of GTP hydrolysis to do work on the phospholipid bilayer. This novel mechanism of membrane fusion is unlike any known fusion protein and establishes a new paradigm. Recent structural work has allowed us to develop a detailed model of atlastin function. We will test important predictions of this model using recombinant proteins, in vitro fusion reactions, measurement of GTPase activity, and determination of oligomeric state. We will probe the specific protein requirements for membrane tethering through the conserved GTPase domain, stable membrane attachment provided by a three helical bundle segment that connects the GTPase domain to transmembrane anchors, and membrane destabilization by an amphipathic helix in the C-terminal cytoplasmic tail. This work will contribute to two very important areas of research, the pathophysiology of Hereditary Spastic Paraplegia and the general mechanism of membrane fusion. Molecular genetic analysis of the most prominent forms of HSP, including atlastin, identified proteins that are generally involved in ER function. Proper functioning of the ER is critical for all cells given the crucial activities this organelle provides with respect to te secretory apparatus, lipid biogenesis, and calcium homeostasis. Recent data suggest that maintenance of a reticular morphology is necessary for ER function and atlastin, in part, provides for the ability to change shape and maintain lumen continuity. Characterization of this new way to merge membranes will be important for understanding the biophysical mechanisms of ER homotypic fusion and ER homeostasis in general. PUBLIC HEALTH RELEVANCE: Hereditary Spastic Paraplegia is an inherited neurological disorder that is characterized by progressive lower extremity weakness and spasticity. Mutations is atlasin-1, account for ~10% of all autosomal dominant forms of the disease. Atlastin is a large GTPase that promotes membrane fusion, specifically of the endoplasmic reticulum. A fundamental understanding of the atlastin's role in generating and maintaining ER function by homotypic ER fusion will significantly inform the mechanistic basis of ER-associated pathologies such as the neuronal degeneration found in Hereditary Spastic Paraplegia.