期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 295, 期 34, 页码 12305-12316出版社
ELSEVIER
DOI: 10.1074/jbc.RA120.013672
关键词
cell biology; trafficking; single particle analysis; membrane bilayer; oligomerization; biochemical reconstitution; retromer; WASHC2C
资金
- NIGMS, National Institutes of Health Grants [R37GM061221, F32GM125120, R01NS113236]
- NINDS, National Institutes of Health [R37GM061221, F32GM125120, R01NS113236]
Retromer orchestrates the selection and export of integral membrane proteins from the endosome via retrograde and plasma membrane recycling pathways. Long-standing hypotheses regarding the retromer sorting mechanism posit that oligomeric interactions between retromer and associated accessory factors on the endosome membrane drives clustering of retromer-bound integral membrane cargo prior to its packaging into a nascent transport carrier. To test this idea, we examined interactions between components of the sorting nexin 3 (SNX3)-retromer sorting pathway using quantitative single particle fluorescence microscopy in a reconstituted system. This system includes a supported lipid bilayer, fluorescently labeled retromer, SNX3, and two model cargo proteins, RAB7, and retromer-binding segments of the WASHC2C subunit of the WASH complex. We found that the distribution of membrane-associated retromer is predominantly comprised of monomer (similar to 18%), dimer (similar to 35%), trimer (similar to 24%), and tetramer (similar to 13%). Unexpectedly, neither the presence of membrane-associated cargo nor accessory factors substantially affected this distribution. The results indicate that retromer has an intrinsic propensity to form low order oligomers on a supported lipid bilayer and that neither membrane association nor accessory factors potentiate oligomerization. The results support a model whereby SNX3-retromer is a minimally concentrative coat protein complex adapted to bulk membrane trafficking from the endosomal system.
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