Fig 2: Complex formation between Ahsa1 and GR requires interaction between Ahsa1 and Hsp90.MDA-MB-231 cells were transfected with pcDNA5/TO plasmids encoding full-length Ahsa1, Ahsa1Δ20, or Ahsa1E67K, each with a C-terminal myc tag. Cells were lysed and Ahsa1 was immunoprecipitated using anti-myc magnetic beads. Total cell lysates and immunoprecipitated material were analyzed with antibodies to Hsp90 and total Ahsa1 or the glucocorticoid hormone receptor and anti-myc antibodies. (A) Hsp90 and (B) glucocorticoid receptor is recovered in complex with full-length Ahsa1 but not Ahsa1Δ20 or Ahsa1E67K (N = 3). Source data are available online for this figure.

Fig 3: Loss of the ICD or ICD and NxNNWHW motif results in a loss of recruitment to Hsp90/client complexes.MDA-MB-231 cells were transfected with pcDNA5/TO plasmids encoding full-length Ahsa1, Ahsa1Δ20, or Ahsa1Δ27, each with a C-terminal myc tag. Cells were lysed and Ahsa1 was immunoprecipitated using anti-myc magnetic beads. Total cell lysates and immunoprecipitated material were analyzed with antibodies to Hsp90 and total Ahsa1 or the glucocorticoid hormone receptor and anti-myc antibodies. (A) Hsp90 and (B) glucocorticoid receptor is recovered in complex with full-length Ahsa1 but not Ahsa1Δ20 or Ahsa1Δ27 (A: N = 5; B: N = 4). Source data are available online for this figure.

Fig 4: The ICD is required for stable interaction with Hsp90 and GR.Band intensity of five replicate experiments was measured by densitometry. After background subtraction, intensities of Hsp90 (A) and GR (B) were normalized against the amount of Ahsa1 recovered and expressed as a percentage of each protein with wildtype Ahsa1. Data Information: In (A), data from five individual experiments are plotted as individual points as well as the mean +/− SD (N = 5). In (B), data from four individual experiments are plotted as individual points as well as the mean +/− SD (N = 4). Statistical significance was calculated for (A, B) using a Dunnett’s multiple comparison’s test (**P ≤ 0.01; ****P ≤ 0.0001). Source data are available online for this figure.

Fig 5: The Ahsa1 ICD modulates ATPase stimulation of Hsp90.ATPase stimulation of Hsp90α and Hsp90β by Ahsa1 constructs (A). Schematic of Ahsa1 constructs used. (B, C) Stimulation of Hsp90α (B) and Hsp90β (C) ATPase activity by increasing concentrations of full-length Ahsa1 (black circles), Ahsa1Δ20 (blue squares), and Ahsa1Δ27 (purple triangles). (D) Bmax values of each of the three replicate reactions shown in (A) blue) and (B) (green). (E) Kapp values of the reactions shown in (A) (blue) and (B) (green). Reactions contained 2 μM Hsp90α or Hsp90β and indicated concentration of cochaperone. N = 3. Data Information: In (B, C), data for each concentration of cochaperone are presented as mean +/− SEM of three independent experiments (N = 3; each N is one experiment carried out with technical triplicates as described in “Methods”). (D, E) The individual Bmax or Kapp values, respectively, are plotted as well as the mean +/− SEM of the three independent experiments shown in (B, C) (N = 3; each N is one experiment carried out with technical triplicates as described in “Methods”). Statistical significance in (D, E) was calculated using a Tukey’s multiple comparisons test (*P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001) (P value symbols in black apply to Hsp90α and Hsp90β; P value symbols in blue apply to Hsp90α; P value symbols in green apply to Hsp90β). Source data are available online for this figure.