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 atl...

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Autores principales:	Kyung Tae Kim, Yeojin Moon, Yunsu Jang, Kang Taek Lee, Changwook Lee, Youngsoo Jun, Sanghwa Lee
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/7b7575a634b2487489396a7293f8fe1c
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spelling	oai:doaj.org-article:7b7575a634b2487489396a7293f8fe1c2021-12-02T12:30:15ZMolecular mechanisms of atlastin-mediated ER membrane fusion revealed by a FRET-based single-vesicle fusion assay10.1038/s41598-017-09162-92045-2322https://doaj.org/article/7b7575a634b2487489396a7293f8fe1c2017-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-09162-9https://doaj.org/toc/2045-2322Abstract 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.Kyung Tae KimYeojin MoonYunsu JangKang Taek LeeChangwook LeeYoungsoo JunSanghwa LeeNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-8 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Kyung Tae Kim Yeojin Moon Yunsu Jang Kang Taek Lee Changwook Lee Youngsoo Jun Sanghwa Lee Molecular mechanisms of atlastin-mediated ER membrane fusion revealed by a FRET-based single-vesicle fusion assay
description	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.
format	article
author	Kyung Tae Kim Yeojin Moon Yunsu Jang Kang Taek Lee Changwook Lee Youngsoo Jun Sanghwa Lee
author_facet	Kyung Tae Kim Yeojin Moon Yunsu Jang Kang Taek Lee Changwook Lee Youngsoo Jun Sanghwa Lee
author_sort	Kyung Tae Kim
title	Molecular mechanisms of atlastin-mediated ER membrane fusion revealed by a FRET-based single-vesicle fusion assay
title_short	Molecular mechanisms of atlastin-mediated ER membrane fusion revealed by a FRET-based single-vesicle fusion assay
title_full	Molecular mechanisms of atlastin-mediated ER membrane fusion revealed by a FRET-based single-vesicle fusion assay
title_fullStr	Molecular mechanisms of atlastin-mediated ER membrane fusion revealed by a FRET-based single-vesicle fusion assay
title_full_unstemmed	Molecular mechanisms of atlastin-mediated ER membrane fusion revealed by a FRET-based single-vesicle fusion assay
title_sort	molecular mechanisms of atlastin-mediated er membrane fusion revealed by a fret-based single-vesicle fusion assay
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/7b7575a634b2487489396a7293f8fe1c
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Molecular mechanisms of atlastin-mediated ER membrane fusion revealed by a FRET-based single-vesicle fusion assay

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