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Sci Rep. 2017 Aug 18;7(1):8700. doi: 10.1038/s41598-017-09162-9.

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

Kim KT1,2,3, Moon Y4,2, Jang Y1,2, Lee KT3, Lee C5, Jun Y6,7, Lee S8,9.

Author information

1
Advanced Photonics Research Institute, Gwangju Institute of Science and Technology, Gwangju, 61005, Republic of Korea.
2
Cell Logistics Research Center, Gwangju Institute of Science and Technology, Gwangju, 61005, Republic of Korea.
3
Department of Chemistry, Gwangju Institute of Science and Technology, Gwangju, 61005, Republic of Korea.
4
School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, 61005, Republic of Korea.
5
Department of Biological Sciences, School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, 44919, Republic of Korea.
6
School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, 61005, Republic of Korea. junys@gist.ac.kr.
7
Cell Logistics Research Center, Gwangju Institute of Science and Technology, Gwangju, 61005, Republic of Korea. junys@gist.ac.kr.
8
Advanced Photonics Research Institute, Gwangju Institute of Science and Technology, Gwangju, 61005, Republic of Korea. sanglee@gist.ac.kr.
9
Cell Logistics Research Center, Gwangju Institute of Science and Technology, Gwangju, 61005, Republic of Korea. sanglee@gist.ac.kr.

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.

PMID:
28821793
PMCID:
PMC5562884
DOI:
10.1038/s41598-017-09162-9
[Indexed for MEDLINE]
Free PMC Article

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