Abstract
Retromer is a protein sorting device that 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 hypothesis, we examined interactions between the components of the SNX3-Retromer sorting pathway using quantitative single particle fluorescence microscopy of a reconstituted system comprising a supported bilayer, Retromer, a model cargo protein, the accessory proteins SNX3, RAB7, and the Retromer-binding segment of the WASHC2C subunit of the WASH complex. The predominant species of membrane associated Retromer are low order: monomers (∼18%), dimers (∼35%), trimers (∼24%) and tetramers (∼24%). Unexpectedly, neither cargo nor accessory factors promote Retromer oligomerization on a supported bilayer. The results indicate that Retromer has an intrinsic propensity to form low order oligomers and that neither membrane association nor accessory factors potentiate oligomerization. Hence, Retromer is a minimally concentrative sorting device adapted to bulk membrane trafficking from the endosomal system.
List of Abbreviations
- AF488
- AlexaFluor488®
- AF546
- AlexaFluor546®
- AF647
- AlexaFluor647®
- DMT
- Divalent Metal Transporter1-II (cargo)
- WASHC2C-21
- Fragment of WASHC2C with all 21 LFa motifs (aa357 to 1318)
- WASHC2C-5
- Fragment of WASHC2C with the final five LFa motifs (aa921 to 1318)
- FRAP
- Fluorescence Recovery After Photobleaching
- LFa
- Leucine-Phenylalanine-acidic residue motif
- NiNTA-DGS
- nickel salt of 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid) succinyl]
- RAB7-GTP
- Rab7-Q67L, a constitutively GTP-bound form of Rab7
- Retromer-RRS
- Retromer with attached Retromer Recognition Sequence (RRS) of DMT cargo
- RhPE
- Rhodamine-phosphatidylethanolamine
- SLB
- Supported lipid bilayer
- SNX3
- Sorting nexin 3
- SNX-BAR
- Sorting nexin-Bin/Amphiphysin/Rvs
- TIRF
- Total Internal Reflection Fluorescence
- VPS35
- Vacuolar Protein Sorting-associated protein 35
- VPS26
- Vacuolar Protein Sorting-associated protein 26
- VPS29
- Vacuolar Protein Sorting-associated protein 29