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Improved reconstitution of yeast vacuole fusion with physiological SNARE concentrations reveals an asymmetric Rab(GTP) requirement
In vitro reconstitution of homotypic yeast vacuole fusion from purified components enables detailed study of membrane fusion mechanisms. Current reconstitutions have yet to faithfully replicate the fusion process in at least three respects: 1) The density of SNARE proteins required for fusion in vit...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The American Society for Cell Biology
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4985260/ https://www.ncbi.nlm.nih.gov/pubmed/27385334 http://dx.doi.org/10.1091/mbc.E16-04-0230 |
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author | Zick, Michael Wickner, William |
author_facet | Zick, Michael Wickner, William |
author_sort | Zick, Michael |
collection | PubMed |
description | In vitro reconstitution of homotypic yeast vacuole fusion from purified components enables detailed study of membrane fusion mechanisms. Current reconstitutions have yet to faithfully replicate the fusion process in at least three respects: 1) The density of SNARE proteins required for fusion in vitro is substantially higher than on the organelle. 2) Substantial lysis accompanies reconstituted fusion. 3) The Rab GTPase Ypt7 is essential in vivo but often dispensable in vitro. Here we report that changes in fatty acyl chain composition dramatically lower the density of SNAREs that are required for fusion. By providing more physiological lipids with a lower phase transition temperature, we achieved efficient fusion with SNARE concentrations as low as on the native organelle. Although fused proteoliposomes became unstable at elevated SNARE concentrations, releasing their content after fusion had occurred, reconstituted proteoliposomes with substantially reduced SNARE concentrations fused without concomitant lysis. The Rab GTPase Ypt7 is essential on both membranes for proteoliposome fusion to occur at these SNARE concentrations. Strikingly, it was only critical for Ypt7 to be GTP loaded on membranes bearing the R-SNARE Nyv1, whereas the bound nucleotide of Ypt7 was irrelevant on membranes bearing the Q-SNAREs Vam3 and Vti1. |
format | Online Article Text |
id | pubmed-4985260 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | The American Society for Cell Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-49852602016-10-30 Improved reconstitution of yeast vacuole fusion with physiological SNARE concentrations reveals an asymmetric Rab(GTP) requirement Zick, Michael Wickner, William Mol Biol Cell Articles In vitro reconstitution of homotypic yeast vacuole fusion from purified components enables detailed study of membrane fusion mechanisms. Current reconstitutions have yet to faithfully replicate the fusion process in at least three respects: 1) The density of SNARE proteins required for fusion in vitro is substantially higher than on the organelle. 2) Substantial lysis accompanies reconstituted fusion. 3) The Rab GTPase Ypt7 is essential in vivo but often dispensable in vitro. Here we report that changes in fatty acyl chain composition dramatically lower the density of SNAREs that are required for fusion. By providing more physiological lipids with a lower phase transition temperature, we achieved efficient fusion with SNARE concentrations as low as on the native organelle. Although fused proteoliposomes became unstable at elevated SNARE concentrations, releasing their content after fusion had occurred, reconstituted proteoliposomes with substantially reduced SNARE concentrations fused without concomitant lysis. The Rab GTPase Ypt7 is essential on both membranes for proteoliposome fusion to occur at these SNARE concentrations. Strikingly, it was only critical for Ypt7 to be GTP loaded on membranes bearing the R-SNARE Nyv1, whereas the bound nucleotide of Ypt7 was irrelevant on membranes bearing the Q-SNAREs Vam3 and Vti1. The American Society for Cell Biology 2016-08-15 /pmc/articles/PMC4985260/ /pubmed/27385334 http://dx.doi.org/10.1091/mbc.E16-04-0230 Text en © 2016 Zick and Wickner. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. |
spellingShingle | Articles Zick, Michael Wickner, William Improved reconstitution of yeast vacuole fusion with physiological SNARE concentrations reveals an asymmetric Rab(GTP) requirement |
title | Improved reconstitution of yeast vacuole fusion with physiological SNARE concentrations reveals an asymmetric Rab(GTP) requirement |
title_full | Improved reconstitution of yeast vacuole fusion with physiological SNARE concentrations reveals an asymmetric Rab(GTP) requirement |
title_fullStr | Improved reconstitution of yeast vacuole fusion with physiological SNARE concentrations reveals an asymmetric Rab(GTP) requirement |
title_full_unstemmed | Improved reconstitution of yeast vacuole fusion with physiological SNARE concentrations reveals an asymmetric Rab(GTP) requirement |
title_short | Improved reconstitution of yeast vacuole fusion with physiological SNARE concentrations reveals an asymmetric Rab(GTP) requirement |
title_sort | improved reconstitution of yeast vacuole fusion with physiological snare concentrations reveals an asymmetric rab(gtp) requirement |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4985260/ https://www.ncbi.nlm.nih.gov/pubmed/27385334 http://dx.doi.org/10.1091/mbc.E16-04-0230 |
work_keys_str_mv | AT zickmichael improvedreconstitutionofyeastvacuolefusionwithphysiologicalsnareconcentrationsrevealsanasymmetricrabgtprequirement AT wicknerwilliam improvedreconstitutionofyeastvacuolefusionwithphysiologicalsnareconcentrationsrevealsanasymmetricrabgtprequirement |