Figure 1.
GST-Apl5-ear binds HOPS subunits. Pulldowns on GST-Apl5-ear resin were performed as described (see Materials and Methods) with ∼150 OD600 nm × ml of BY4742 yeast detergent lysate. Bounds proteins were eluted by incubating beads in 20 mM Tris-Cl, 20 mM glutathione, and 600 mM NaCl for 10 min at 4°C (top panel) or SDS loading buffer and heating at 95°C for 5 min (bottom panel). Twenty percent of the load (L) and 20% of the unbound fraction (U) relative to the bound fraction (B) were loaded, separated by SDS-PAGE, and analyzed by immunoblot. Asterisk (*) indicates nonspecific bands detected by the antibodies. The various forms of ALP are indicated: p, proALP; m, mature ALP; and s, soluble ALP. In wild-type cells, a large majority of ALP exists in the mature form.