Figure 4.

Effects of Bag2 mutations on Hsc70 binding, nucleotide exchange and substrate refolding activity. (a) GST-pulldown assays using wild-type or mutant GST-tagged human Bag2. Hsc70-NBD was incubated with GST-Bag2 prebound to GST-minicolumns, which were subsequently washed and eluted with 10 mM glutathione. Eluted proteins were resolved using SDS-PAGE and stained with Coomassie blue. Symbols above lanes qualitatively designate the amount of Hsc70-NBD bound to and co-eluting with GST-Bag2 (++: strong binding, +: weak binding; −: no binding). In lane “GST”, the GST protein is not within the portion of the gel shown in the figure. Positions of 50 and 37 kD molecular weight markers are shown at right. (b) Single-turnover nucleotide exchange measurements on Hsc70. Time course of α-³²P-ATP release from Hsc70 in the absence (black circles) or presence of wild-type Bag2 (red squares) or Bag2 mutants. Data shown are the means of three independent experiments. (c) Effect of Bag2 on refolding of heat-denatured luciferase by Hsc70/Hsp40. “HD Luc” designates background luminescence signal from heat-denatured luciferase alone, without any chaperones present. “Hsc70 only” designates signal from Hsc70 without Hsp40 present. “HsBag2-Mut” corresponds to the human Bag2-Q153A/Q156A/Q167A/K168A/K171A mutant. Hs and Mm designate human and murine Bag2 constructs respectively. All of the experiments containing Bag2 constructs also contained 1 μM Hsc70 and 1 μM Hsp40. Signal from non-denatured luciferase incubated for equivalent amounts of time in refolding buffer was set to 100%. (d) Concentration dependence of promotion of heat-denatured luciferase refolding by Bag2. All experiments also contained 1 μM Hsc70 and 1 μM Hsp40. Luciferase signal was recorded 1 hour after initiation of refolding reaction. Bovine serum albumin (BSA) was used as a negative control. Signal from non-denatured luciferase incubated for equivalent amounts of time in refolding buffer was set to 100%.