The biogenesis and maintenance of the endoplasmic reticulum (ER) requires membrane fusion. ER homotypic fusion is driven by the large GTPase atlastin. Domain analysis of atlastin shows that a conserved region of the C-terminal cytoplasmic tail is absolutely required for fusion activity. Atlastin in adjacent membranes must associate to bring the ER membranes into molecular contact. Drosophila atlastin dimerizes in the presence of GTPγS but is monomeric with GDP or without nucleotide. Oligomerization requires the juxtamembrane middle domain three-helix bundle, as does efficient GTPase activity. A soluble version of the N-terminal cytoplasmic domain that contains the GTPase domain and the middle domain three-helix bundle serves as a potent, concentrationdependent inhibitor of membrane fusion both in vitro and in vivo. However, atlastin domains lacking the middle domain are without effect. GTP-dependent dimerization of atlastin generates an enzymatically active protein that drives membrane fusion after nucleotide hydrolysis and conformational reorganization. M embrane fusion reactions are 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. SNARE proteins are responsible for membrane fusion within the secretory pathway as well as homotypic fusion of endosomes and lysosomes (1, 2). The fusion of other organelles such as mitochondria and the endoplasmic reticulum (ER) are less well characterized. Many observations suggest that mitochondrial fusion is driven by proteins called mitofusins (3-5). Although ER-resident SNARE proteins are required for vesicular transport back to the ER (6, 7), the protein(s) responsible for the generation and maintenance of the ER has only recently been discovered. We have recently shown that Atlastin is a GTP-dependent membrane fusion protein that is responsible for ER homotypic fusion (8). Membrane fusion provided by atlastin helps shape and maintain the dynamic nature of the ER membrane tubule network (9-13).Atlastin is the product of the human SPG3A locus (14). SPG3A (Atl1) is a member of a larger family of genes that are responsible for a group of inherited neurological disorders called hereditary spastic paraplegia (HSP) (15,16). This disease is characterized by progressive lower-extremity weakness and spasticity. The neuropathological basis for compromised motor function in HSP is likely length-dependent axonopathy of the corticospinal tract (16). Twenty HSP gene products have been identified, and their molecular analysis has suggested that three general categories of proteins may be responsible for HSP. These gene functions fall into three broad groups, including intracellular trafficking, mitochondrial function, and axonal pathfinding and myelination (17). More than half of all HSP cases are caused by mutation in ER-resident or ER-associated proteins (18)(19)(20). Atlastin and the ER tubule-forming protein receptor expression-enhancing prot...

The mechanisms governing atlastin-mediated membrane fusion are unknown. Here we demonstrate that a three-helix bundle (3HB) within the middle domain is required for oligomerization. Mutation of core hydrophobic residues within these helices inactivates atlastin function by preventing membrane tethering and the subsequent fusion. GTP binding induces a conformational change that reorients the GTPase domain relative to the 3HB to permit self-association, but the ability to hydrolyze GTP is required for full fusion, indicating that nucleotide binding and hydrolysis play distinct roles. Oligomerization of atlastin stimulates its ability to hydrolyze GTP, and the energy released drives lipid bilayer merger. Mutations that prevent atlastin self-association also abolish oligomerization-dependent stimulation of GTPase activity. Furthermore, increasing the distance of atlastin complex formation from the membrane inhibits fusion, suggesting that this distance is crucial for atlastin to promote fusion.Drosophila | endoplasmic reticulum T he endoplasmic reticulum (ER) forms an elaborate network that spreads throughout the cell. The ER is a dynamic organelle, continuously undergoing membrane fusion. One of the primary functions of the ER is folding and glycosylation of secreted proteins, as well as the distribution of resident membrane proteins within the secretory pathway. Vesicular traffic exiting and entering the ER requires heterotypic membrane fusion, which uses the SNARE protein family and their associated chaperones [soluble N-ethylmaleimide-sensitive factor attachment protein receptors] for membrane merger (1). In contrast to vesicular transport, the establishment and maintenance of the ER network requires homotypic membrane fusion (2, 3). Membrane fusion occurs through an initial tethering step, which locks apposing membranes together, followed by lipid bilayer merger. Drosophila atlastin forms transoligomeric complexes between adjacent ER membranes and promotes liposome fusion in vitro, and its overexpression induces ER fusion in vivo, indicating that this GTPase is responsible for mediating ER homotypic fusion (4).The atlastins constitute a family of very closely related, integral membrane GTPases. They are distant members of the dynamin family of GTPases and are localized on the ER membrane. Mammals have three atlastins, and mutations in ATL1 are responsible for one of the most frequent and earliest-onset forms of pure hereditary spastic paraplegia (5, 6). Human atlastins interact with the ER tubule-shaping proteins reticulons and DP1 and have been proposed to play a role in the formation of an interconnected tubular network, indirectly implicating these GTPases in the fusion of ER membranes (7). Structurally atlastins resemble mitofusins, a class of GTPases also belonging to the dynamin family essential for the homotypic fusion of mitochondria membranes (8, 9), although the ability of mitofusins to directly induce lipid bilayer merger has yet to be demonstrated (10).Despite the identification of the dual tetherin...