Figure 2.
GST-Apl5-ear interacts with the HOPS holocomplex. (A) Cell lysate from BJ3505 GFP-Vps39 was incubated for 1 h with GST-Apl5-ear glutathione resin. The resin was washed, and interacting proteins were eluted by a linear gradient of 0–0.7 M NaCl. Fractions were immunoblotted for the presence of HOPS. (B) Peak HOPS fractions were pooled, and 500 μl of the pooled peak was subjected to immunoprecipitation by α-GFP antibodies immobilized on Protein A-Sepharose. Note that the unbound material is depleted of all HOPS subunits but not of Vam7, which binds to AP-3 independently of HOPS. (C) The pooled HOPS peak was also subjected to size exclusion chromatography on a Superose 6 column. In calibration runs, blue dextran (2 MDa) eluted at fraction 17 (not shown), and thyroglobulin (670 kDa) eluted at fraction 26, as indicated. The exclusion limit of Superose 6 is Mr ∼4 × 107.