Molecular mechanisms of atlastin-mediated ER membrane fusion revealed by a FRET-based single-vesicle fusion assay

Abstract

Homotypic fusion of endoplasmic reticulum membranes is driven by atlastin GTPases; however, the underlying mechanism remains largely unknown. Here, using a FRET-based single-vesicle fusion assay with liposomes bearing the yeast atlastin Sey1p, we investigated the molecular mechanisms of atlastin-mediated membrane tethering and fusion. Although Sey1p-bearing proteoliposomes frequently underwent membrane tethering in a GTP hydrolysis-dependent manner as reported in studies using bulk assays, only a small fraction of the tethered liposomes proceeded to fusion. Strikingly, the rest of the tethered liposomes failed to fuse or dissociate. This stable tethering, however, did not require continued GTP hydrolysis because GTP omission and magnesium chelation did not disrupt tethering. Interestingly, an increased Sey1p density on the membrane markedly accelerated tethering but barely affected the fusion rate of the tethered liposomes, indicating that Sey1p requires additional factors to support efficient fusion in vivo. Finally, the assay also revealed that Sey1p-mediated liposome fusion occurs through hemifusion, suggesting the mechanistic conservation between biological membrane fusion events despite the existence of diverse fusogens.

Ergosterol interacts with Sey1p to promote atlastin‐mediated endoplasmic reticulum membrane fusion inSaccharomyces cerevisiae

“…Large unilamellar vesicles were formed by 5 freeze-thaw cycles in liquid N 2 and water at 37°C and then extruded 11 times through a polycarbonate filter (Avanti Polar Lipids) with a pore size of 100 nm to form unilamellar liposomes of a uniform size. Sey1p reconstitution was performed by detergent-assisted insertion as previously described (17,26). In brief, purified Sey1p prepared in 0.1% Triton X-100 was mixed with preformed liposomes at a protein-to-lipid ratio of 1:1000 and an effective detergent-tolipid ratio (R eff ) of ,0.64 and incubated at 4°C for 2 h. R eff is defined by the following equation: R eff = (D total 2 D water )/ (total lipid), where D total is the total detergent concentration and D water is the monomeric detergent concentration (0.18 mM for Triton X-100).…”

Dictyosteliumlacking the single atlastin homolog Sey1 shows aberrantERarchitecture, proteolytic processes and expansion of theLegionella‐containing vacuole

“…Structural and biochemical studies indicate that upon GTP binding, the GTPase domains of ATL from two distinct ER tubules can form trans‐homodimers, thus promoting membrane contact and fusion (Bian et al, 2011; Byrnes & Sondermann, 2011). The fusogenic properties of ATLs have been molecularly detailed by in vitro membrane fusion studies (Anwar et al, 2012; Bian et al, 2011; Kim et al, 2017; Orso et al, 2009) and crystal structure analysis (Bian et al, 2011; Byrnes et al, 2013; Byrnes & Sondermann, 2011; Liu et al, 2012, 2015). In addition to ER dynamics, ATLs contribute to other cellular processes including lipid droplet biogenesis (Klemm et al, 2013), mitochondrial lipid exchange (Voss, Lahiri, Young, Loewen, & Prinz, 2012), membrane tethering (Krols et al, 2018; Niu et al, 2019), endosomal transport (Stefano et al, 2015), inner nuclear membrane protein insertion (Pawar, Ungricht, Tiefenboeck, Leroux, & Kutay, 2017), and selective autophagy (Q. Chen et al, 2019; Liang, Lingeman, Ahmed, & Corn, 2018).…”

A disease causing ATLASTIN 3 mutation affects multiple endoplasmic reticulum-related pathways

“…ATLs can dimerize in cis and trans, and are believed to tether adjacent membranes and bring them closely together to allow fusion. ATLs can mediate fusion in vitro, but for efficient fusion additional factors were proposed [ 14 ]. Mammalian ATLs differ in tissue expression, with ATL1 being mainly expressed in the CNS [ 8 , 15 ] whereas ATL2 and ATL3 are more ubiquitously expressed [ 16 ].…”