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.
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We thank J. Lippincott-Schwartz, R. Rikhy, M. Buszczak, M. Ramaswami and J. Kim for providing Drosophila strains, A. Gazziero for generating transgenic lines, and E. Giordano for the provision of the SympUAST vector. We thank M. G. Rossetto for support throughout the duration of the work, N. D’Elia for technical assistance, R. Polishchuck and the Telethon EM core facility, and W. Guo for help with EM analysis of negative stained Drosophila Atlastin proteoliposomes. This work was supported by grants from the National Institutes of Health (GM71832) and the G. Harold and Leila Mathers Charitable Foundation to J.A.M., and from Telethon-Italy, the Italian Ministry of Health and the Foundation Compagnia di San Paolo to A.D.
Author Contributions G.O. and D.P. contributed to the experimental design and carried out all Drosophila work. D.P. developed and performed the vesicle immunoprecipitation assay. J.T. performed HeLa cell transfection and immunoprecipitation experiments. S.L., T.J.M. and J.E.F. carried out liposome production, analyses and in vitro fusion. M.M. and A.E. conducted electron microscopy experiments. A.M. contributed to the experimental design and discussions. J.A.M. designed and supervised liposome production, analyses and in vitro fusion and contributed to writing the manuscript. A.D. conceived and designed the study, supervised Drosophila and cell culture experiments and wrote the manuscript.
This file contains Supplementary Notes, Supplementary Figures S1-S16 with Legends and Supplementary References. (PDF 4738 kb)
This movie shows a 3D reconstruction of confocal stacks from wild type muscle labelled with GFP-KDEL. (MPG 5698 kb)
This movie shows a 3D reconstruction of confocal stacks from Datlastin mutant muscle labelled with GFP-KDEL. Loss of Datlastin results in a more sparse and fragmented ER network compared to wild type animal in movie 1. (MPG 5266 kb)
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Orso, G., Pendin, D., Liu, S. et al. Homotypic fusion of ER membranes requires the dynamin-like GTPase Atlastin. Nature 460, 978–983 (2009). https://doi.org/10.1038/nature08280
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