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Quantitative high-content imaging identifies novel regulators of Neo1 trafficking at endosomes

P4-ATPases are a family of putative phospholipid flippases that regulate lipid membrane asymmetry, which is important for vesicle formation. Two yeast flippases, Drs2 and Neo1, have nonredundant functions in the recycling of the synaptobrevin-like v-SNARE Snc1 from early endosomes. Drs2 activity is...

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Autores principales: Dalton, Lauren E., Bean, Björn D. M., Davey, Michael, Conibear, Elizabeth
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The American Society for Cell Biology 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5449152/
https://www.ncbi.nlm.nih.gov/pubmed/28404745
http://dx.doi.org/10.1091/mbc.E16-11-0772
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author Dalton, Lauren E.
Bean, Björn D. M.
Davey, Michael
Conibear, Elizabeth
author_facet Dalton, Lauren E.
Bean, Björn D. M.
Davey, Michael
Conibear, Elizabeth
author_sort Dalton, Lauren E.
collection PubMed
description P4-ATPases are a family of putative phospholipid flippases that regulate lipid membrane asymmetry, which is important for vesicle formation. Two yeast flippases, Drs2 and Neo1, have nonredundant functions in the recycling of the synaptobrevin-like v-SNARE Snc1 from early endosomes. Drs2 activity is needed to form vesicles and regulate its own trafficking, suggesting that flippase activity and localization are linked. However, the role of Neo1 in endosomal recycling is not well characterized. To identify novel regulators of Neo1 trafficking and activity at endosomes, we first identified mutants with impaired recycling of a Snc1-based reporter and subsequently used high-content microscopy to classify these mutants based on the localization of Neo1 or its binding partners, Mon2 and Dop1. This analysis identified a role for Arl1 in stabilizing the Mon2/Dop1 complex and uncovered a new function for Vps13 in early endosome recycling and Neo1 localization. We further showed that the cargo-selective sorting nexin Snx3 is required for Neo1 trafficking and identified an Snx3 sorting motif in the Neo1 N-terminus. Of importance, the Snx3-dependent sorting of Neo1 was required for the correct sorting of another Snx3 cargo protein, suggesting that the incorporation of Neo1 into recycling tubules may influence their formation.
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spelling pubmed-54491522017-08-16 Quantitative high-content imaging identifies novel regulators of Neo1 trafficking at endosomes Dalton, Lauren E. Bean, Björn D. M. Davey, Michael Conibear, Elizabeth Mol Biol Cell Articles P4-ATPases are a family of putative phospholipid flippases that regulate lipid membrane asymmetry, which is important for vesicle formation. Two yeast flippases, Drs2 and Neo1, have nonredundant functions in the recycling of the synaptobrevin-like v-SNARE Snc1 from early endosomes. Drs2 activity is needed to form vesicles and regulate its own trafficking, suggesting that flippase activity and localization are linked. However, the role of Neo1 in endosomal recycling is not well characterized. To identify novel regulators of Neo1 trafficking and activity at endosomes, we first identified mutants with impaired recycling of a Snc1-based reporter and subsequently used high-content microscopy to classify these mutants based on the localization of Neo1 or its binding partners, Mon2 and Dop1. This analysis identified a role for Arl1 in stabilizing the Mon2/Dop1 complex and uncovered a new function for Vps13 in early endosome recycling and Neo1 localization. We further showed that the cargo-selective sorting nexin Snx3 is required for Neo1 trafficking and identified an Snx3 sorting motif in the Neo1 N-terminus. Of importance, the Snx3-dependent sorting of Neo1 was required for the correct sorting of another Snx3 cargo protein, suggesting that the incorporation of Neo1 into recycling tubules may influence their formation. The American Society for Cell Biology 2017-06-01 /pmc/articles/PMC5449152/ /pubmed/28404745 http://dx.doi.org/10.1091/mbc.E16-11-0772 Text en © 2017 Dalton et al. 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
Dalton, Lauren E.
Bean, Björn D. M.
Davey, Michael
Conibear, Elizabeth
Quantitative high-content imaging identifies novel regulators of Neo1 trafficking at endosomes
title Quantitative high-content imaging identifies novel regulators of Neo1 trafficking at endosomes
title_full Quantitative high-content imaging identifies novel regulators of Neo1 trafficking at endosomes
title_fullStr Quantitative high-content imaging identifies novel regulators of Neo1 trafficking at endosomes
title_full_unstemmed Quantitative high-content imaging identifies novel regulators of Neo1 trafficking at endosomes
title_short Quantitative high-content imaging identifies novel regulators of Neo1 trafficking at endosomes
title_sort quantitative high-content imaging identifies novel regulators of neo1 trafficking at endosomes
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5449152/
https://www.ncbi.nlm.nih.gov/pubmed/28404745
http://dx.doi.org/10.1091/mbc.E16-11-0772
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