Subjects: Biology >> Biophysics >> Cell Biology submitted time 2016-05-11
Yan, Liming
Wang, Wei
Rao, Zihe
Lou, Zhiyong
Sun, Sha
Shi, Juanming
Hu, Xiaoyu
Hu, Junjie
Sun, Sha
Shi, Juanming
Hu, Xiaoyu
Hu, Junjie
Rao, Zihe
Hu, Junjie
Wang, Wei
Wang, Shiyan
Su, Dan
Su, Dan
Rao, Zihe
Lou, Zhiyong
Abstract： Homotypic membrane fusion of the endoplasmic reticulum is mediated by dynamin-like guanosine triphosphatases (GTPases), which include atlastin (ATL) in metazoans and Sey1p in yeast. In this paper, we determined the crystal structures of the cytosolic domain of Sey1p derived from Candida albicans. The structures reveal a stalk-like, helical bundle domain following the GTPase, which represents a previously unidentified configuration of the dynamin superfamily. This domain is significantly longer than that of ATL and critical for fusion. Sey1p forms a side-by-side dimer in complex with GMP-PNP or GDP/AlF4- but is monomeric with GDP. Surprisingly, Sey1p could mediate fusion without GTP hydrolysis, even though fusion was much more efficient with GTP. Sey1p was able to replace ATL in mammalian cells, and the punctate localization of Sey1p was dependent on its GTPase activity. Despite the common function of fusogenic GTPases, our results reveal unique features of Sey1p.
Peer Review Status:
Awaiting Review
Subjects: Biology >> Biophysics >> Cell Biology submitted time 2016-05-11
Wu, Fuyun
Hu, Xiaoyu
Bian, Xin
Hu, Junjie
Wu, Fuyun
Hu, Xiaoyu
Bian, Xin
Hu, Junjie
Liu, Xinqi
Liu, Xinqi
Hu, Junjie
Abstract： Formation of the endoplasmic reticulum (ER) network requires homotypic membrane fusion, which involves a class of atlastin (ATL) GTPases. Purified Drosophila ATL is capable of mediating vesicle fusion in vitro, but such activity has not been reported for any other ATLs. Here, we determined the preliminary crystal structure of the cytosolic segment of Drosophila ATL in a GDP-bound state. The structure reveals a GTPase domain dimer with the subsequent three-helix bundles associating with their own GTPase domains and pointing in opposite directions. This conformation is similar to that of human ATL1, to which GDP and high concentrations of inorganic phosphate, but not GDP only, were included. Drosophila ATL restored ER morphology defects in mammalian cells lacking ATLs, and measurements of nucleotide-dependent dimerization and GTPase activity were comparable for Drosophila ATL and human ATL1. However, purified and reconstituted human ATL1 exhibited no in vitro fusion activity. When the cytosolic segment of human ATL1 was connected to the transmembrane (TM) region and C-terminal tail (CT) of Drosophila ATL, the chimera still exhibited no fusion activity, though its GTPase activity was normal. These results suggest that GDP-bound ATLs may adopt multiple conformations and the in vitro fusion activity of ATL cannot be achieved by a simple collection of functional domains.
Peer Review Status:Awaiting Review
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