Establishment and maintenance of proper architecture is essential for endoplasmic reticulum (ER) function. Homotypic membrane fusion is required for ER biogenesis and maintenance, and has been shown to depend on GTP hydrolysis. Here we demonstrate that Drosophila Atlastin--the fly homologue of the mammalian GTPase atlastin 1 involved in hereditary spastic paraplegia--localizes on ER membranes and that its loss causes ER fragmentation. Drosophila Atlastin embedded in distinct membranes has the ability to form trans-oligomeric complexes and its overexpression induces enlargement of ER profiles, consistent with excessive fusion of ER membranes. In vitro experiments confirm that Atlastin autonomously drives membrane fusion in a GTP-dependent fashion. In contrast, GTPase-deficient Atlastin is inactive, unable to form trans-oligomeric complexes owing to failure to self-associate, and incapable of promoting fusion in vitro. These results demonstrate that Atlastin mediates membrane tethering and fusion and strongly suggest that it is the GTPase activity that is required for ER homotypic fusion.
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...
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