RESUMEN
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.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/enzimología , Dinaminas , Retículo Endoplásmico/metabolismo , GTP Fosfohidrolasas/metabolismo , Fusión de Membrana , Animales , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Retículo Endoplásmico/patología , GTP Fosfohidrolasas/deficiencia , GTP Fosfohidrolasas/genética , Células HeLa , Humanos , Transporte de Proteínas , Proteolípidos/metabolismoRESUMEN
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.