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

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-med...

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Bibliographic Details
Published in:Scientific reports 2017-08, Vol.7 (1), p.8700-8, Article 8700
Main Authors: Kim, Kyung Tae, Moon, Yeojin, Jang, Yunsu, Lee, Kang Taek, Lee, Changwook, Jun, Youngsoo, Lee, Sanghwa
Format: Article
Language:English
Subjects:
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631/57/2265
631/80/313/2378
82/80
82/83
Biological Assay - methods
Biological membranes
Chelation
Efficiency
Endoplasmic reticulum
Endoplasmic Reticulum - metabolism
Fluorescence Resonance Energy Transfer
Guanosine triphosphate
Guanosine Triphosphate - metabolism
Humanities and Social Sciences
Hydrolysis
Life sciences
Lipids
Liposomes
Magnesium
Membrane Fusion
Membranes
Molecular modelling
multidisciplinary
Polyethylene glycol
Proteins
Real time
Saccharomyces cerevisiae Proteins - metabolism
Science
Science (multidisciplinary)
Vesicle fusion
Vesicular Transport Proteins - metabolism
Yeasts
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Summary: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.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-017-09162-9