FIGURE 1:

Purification of retromer subcomplexes. (A) Model of Ypt7 interaction with retromer subcomplexes and HOPS on endosomes and vacuoles. 7, Ypt7; CSC, cargo selection complex; SNX, sorting nexin BAR complex. (B–D) Analysis of purified retromer subcomplex. (B) Overproduced CSC and SNX-BAR complexes were purified via the C-terminal TAP tag on Vps35 (CSC) or the SNX subunit Vps5. SNX-GFP had in addition a C-terminal GFP on Vps17; a CSC-GFP Vps29 was tagged similarly. Complexes were analyzed via SDS–PAGE, and gels were stained with Coomassie. (C, D) Gel filtration of SNX-BAR and CSC. TEV eluates from IgG columns were applied to gel filtration as described in Materials and Methods. TEV indicates the migration of the tobacco etch virus protease used to elute the protein from IgG beads. (E) Interaction of purified CSC and SNX-BAR with Rabs. C-terminal GST-tagged Vps21 and Ypt7 Rabs charged with either GDP or GTPγS were immobilized on GSH beads and incubated with purified SNX-BAR and CSC complexes. Eluted proteins were analyzed by SDS–PAGE and Western blotting with antibodies against the calmodulin-binding peptide (cbp; top of displayed gel) or directly analyzed by Coomassie staining (bottom). (F, G) Effect of purified CSC complex on vacuole fusion. Purified vacuoles from the two tester strains were incubated in the presence of ATP for 90 min at 26°C in the presence of the indicated amount of purified CSC (F). In G, the nonspecific GAP Gyp1-46 and the Ypt7 GEF Mon1-Ccz were included in the vacuole fusion reaction. Fusion reactions contained either no CSC or two different amounts of inhibitory CSC concentrations based on the titration in F. HOPS was titrated into each assay as indicated, and fusion was assayed after 90 min at 26°C (see Materials and Methods).