Homotypic membrane fusion of the endoplasmic reticulum is mediated by dynamin-like guanosine triphosphatases

The ER network consists of a continuous membrane system of tubules and sheets (Shibata et al., 2006). ER membranes are interconnected by homotypic fusion to maintain a reticular pattern and proper functioning. In metazoans, fusion is mediated by a class of dynamin-like GTPases called atlastin (ATL; Hu et al., 2009; Orso et al., 2009). In other eukaryotic organisms lacking ATL, a similar class of GTPases, including Sey1p in Saccharomyces cerevisiae (Hu et al., 2009; Anwar et al., 2012) and RHD3 in Arabidopsis thaliana (Zhang et al., 2013), has been identified. Deletion of Sey1p or depletion of ATL proteins in cells results in unbranched ER (Hu et al., 2009), delayed ER fusion (Anwar et al., 2012), or even ER fragmentation (Orso et al., 2009). When purified and reconstituted in vitro, members of both classes have been shown to mediate membrane fusion (Orso et al., 2009; Bian et al., 2011; Anwar et al., 2012; Zhang et al., 2013). GTPase-based ER fusogens play critical physiological roles. In Candida albicans, deletion of Sey1p results in decreased virulence (Yamada-Okabe and Yamada-Okabe, 2002). In A. thaliana, lack of RHD3 leads to short and wavy root hairs and cell expansion defects (Schiefelbein and Somerville, 1990; Chen et al., 2011; Stefano et al., 2012), and the deletion of another family member causes lethality (Zhang et al., 2013). In Drosophila melanogaster and Danio rerio, depletion of ATL causes neuronal defects (Lee et al., 2008, 2009). In humans, mutations in ATL1, the dominant form of ATL in the central nervous system, are tightly associated with hereditary spastic paraplegia, a neurodegenerative disease characterized by axon shortening in corticospinal motor neurons and progressive spasticity and weakness of the lower limbs (Zhao et al., 2001; Salinas et al., 2008). ATL, Sey1p, and RHD3 are anchored in the ER membrane by two closely spaced transmembrane (TM) segments, exposing both termini to the cytosol. The mechanism for homotypic ER fusion has been partially unveiled using ATLs (Hu et al., 2011; Lin et al., 2012). It is very different from SNARE or viral protein-mediated fusion. Recent structural and biochemical studies indicate that the GTPase domain of ATL forms a dimer upon GTP binding, initiating contact between apposing membranes (Bian et al., 2011; Byrnes and Sondermann, 2011). The region following the GTPase domain is a three-helix bundle (3HB) and can dock to its own GTPase domain or that of a neighboring molecule, undergoing major conformational changes during the GTP cycle to drive fusion (Bian et al., 2011; Homotypic membrane fusion of the endoplasmic reticulum is mediated by dynamin-like guanosine triphosphatases (GTP ases), 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. Structures of the yeast dynamin-like GTPase Sey1p provide insight into homotypic ER fusion