Retromer forms low order oligomers on supported lipid bilayers.

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 (~18%), dimer (~35%), trimer (~24%) and tetramer (~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.