Fig. 3.

Digestion products of rSsa2p are substantially altered by BHst 5 binding. Purified rSsa2p (10 μg) was incubated with 20 μg of BHst 5 in the presence of 2 mM ATP-Mg²⁺ and cross-linker for 30 min at room temperature. Stabilized rSsa2p–BHst 5 complexes were isolated by SA beads pull-down. The complexes was then subjected to 5 μg ml⁻¹ thermolysin digestion at 30°C for 1 h. Digestion fragments not associated with the complex binding site were removed by washing, and the remaining bead-retained rSsa2p–BHst 5 complex was recovered and half was subjected to 15% SDS-PAGE and detected by silver staining (lane 4). Ssa2p digestion products differed quite substantially in the presence of Hst 5. Two peptides within the ATPase domain were identified by MS analysis of a digestion fragment (arrow). Lane 1: rSsa2p (0.5 μg); lane 2: thermolysin (0.5 μg); lane 3: thermolysin digestion of rSsa2p without BHst 5; lane 4: thermolysin digestion products from rSsa2p–BHst 5 complex.