Assembling Retromer-coated membrane tubules for biochemical and structural studies

The evolutionarily conserved Retromer complex, composed of Vps29, Vps26 and Vps35, is an essential regulator of endosomal retrieval of transmembrane cargo proteins. For cargo sorting and trafficking to take place, Retromer assembles into coated tubulovesicular carriers together with various sorting nexin (SNX) adaptor proteins including SNX3 and SNX27 in metazoans, and Snx3 or the dimeric Vps5-Vps17 SNX-BAR proteins in yeast. Although Retromer-coated tubulovesicular carriers are vital for its function, the in vitro reconstitution of these membrane assemblies for structural and functional studies can be technically challenging. Approaches include the use of giant unilamellar vesicles and supported membrane tubules for fluorescence imaging, or smaller multilamellar vesicles (MLVs) to generate uniform tubules for imaging by cryoelectron tomography (CryoET). This chapter describes protocols for producing MLVs for membrane binding studies of Retromer and assembling the yeast Retromer-Vps5-Vps17 heteropentameric complex for reconstituting membrane tubulation for CryoET studies. We also discuss our observations of both poorly ordered and well-ordered Retromer coats observed in this experimental setup.