Molecular mechanisms of atlastin-mediated ER membrane fusion revealed by a FRET-based single-vesicle fusion assay
Autor: | Youngsoo Jun, Kang Taek Lee, Yeojin Moon, Yunsu Jang, Changwook Lee, Kyung Tae Kim, Sanghwa Lee |
---|---|
Jazyk: | angličtina |
Rok vydání: | 2017 |
Předmět: |
0301 basic medicine
Atlastin Vesicle fusion Saccharomyces cerevisiae Proteins GTP' Science Vesicular Transport Proteins GTPase Biology Endoplasmic Reticulum Membrane Fusion Article 03 medical and health sciences Fluorescence Resonance Energy Transfer Liposome Multidisciplinary Endoplasmic reticulum Hydrolysis Lipid bilayer fusion Biological membrane 030104 developmental biology Biochemistry Biophysics Medicine Biological Assay Guanosine Triphosphate |
Zdroj: | Scientific Reports SCIENTIFIC REPORTS(7) Scientific Reports, Vol 7, Iss 1, Pp 1-8 (2017) |
ISSN: | 2045-2322 |
Popis: | 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. |
Databáze: | OpenAIRE |
Externí odkaz: |