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Mol Biol Cell. 2017 Jun 1;28(11):1539-1550. doi: 10.1091/mbc.E16-11-0772. Epub 2017 Apr 12.

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

Author information

1
Centre for Molecular Medicine and Therapeutics, BC Children's Hospital, University of British Columbia, Vancouver, BC V5Z 4H4, Canada.
2
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
3
Centre for Molecular Medicine and Therapeutics, BC Children's Hospital, University of British Columbia, Vancouver, BC V5Z 4H4, Canada conibear@cmmt.ubc.ca.
4
Department of Medical Genetics, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.

Abstract

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.

PMID:
28404745
PMCID:
PMC5449152
DOI:
10.1091/mbc.E16-11-0772
[Indexed for MEDLINE]
Free PMC Article

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