RT Journal Article
SR Electronic
T1 Retromer forms low order oligomers on supported lipid bilayers
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.03.31.018150
DO 10.1101/2020.03.31.018150
A1 Catherine L. Deatherage
A1 Joerg Nikolaus
A1 Erdem Karatekin
A1 Christopher G. Burd
YR 2020
UL http://biorxiv.org/content/early/2020/03/31/2020.03.31.018150.abstract
AB 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.AF488AlexaFluor488®AF546AlexaFluor546®AF647AlexaFluor647®DMTDivalent Metal Transporter1-II (cargo)WASHC2C-21Fragment of WASHC2C with all 21 LFa motifs (aa357 to 1318)WASHC2C-5Fragment of WASHC2C with the final five LFa motifs (aa921 to 1318)FRAPFluorescence Recovery After PhotobleachingLFaLeucine-Phenylalanine-acidic residue motifNiNTA-DGSnickel salt of 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid) succinyl]RAB7-GTPRab7-Q67L, a constitutively GTP-bound form of Rab7Retromer-RRSRetromer with attached Retromer Recognition Sequence (RRS) of DMT cargoRhPERhodamine-phosphatidylethanolamineSLBSupported lipid bilayerSNX3Sorting nexin 3SNX-BARSorting nexin-Bin/Amphiphysin/RvsTIRFTotal Internal Reflection FluorescenceVPS35Vacuolar Protein Sorting-associated protein 35VPS26Vacuolar Protein Sorting-associated protein 26VPS29Vacuolar Protein Sorting-associated protein 29