Figure 3.
An intact holocomplex is required for efficient Apl5 binding by HOPS subunits. Pulldowns on GST-Apl5-ear were performed using detergent lysate from BY4742 (WT) and the deletion mutants (A) vps39Δ and vps41Δ, (B) vps16Δ and vps8Δ, or (C) ypt7Δ as described for Figure 1. Interacting proteins were eluted by incubating beads twice in 20 mM Tris-Cl, pH 7.9, 20 mM glutathione, 600 mM NaCl, and 1% Triton X-100 for 10 min at 4°C. The load (L) and unbound (U) fractions correspond to 20% of the bound (B) fraction. Asterisk (*) indicates a nonspecific band detected by the Ypt7 antibody. Note that the Vps16 antibody also detects a nonspecific band that is enriched on the GST-Apl5 resin but not the GST resin, as seen in the vps16Δ eluates, resulting in a higher apparent Vps16 signal. (D) AP-3 preferentially binds Vps41 as part of the HOPS complex. The organization of the HOPS subunits is a composite model from yeast two-hybrid assays and immunoprecipitation from the Merz Lab (unpublished results) as well as published literature (Rieder et al., 1997; Wurmser et al., 2000). Although Vps41 interacts with the remaining HOPS subunits in the absence of Vps39 (unpublished results), the exact subunit interactions have not been mapped.