Figure 4. V domain stimulates Vps4... | Rockefeller University Press

Figure 4.

V domain stimulates Vps4 ATPase activity in vitro. (A) GST-Bro1 fragments and GST-bound beads were incubated with His6-Vps4. Bound material was visualized by both EZBiolab Instant-Band protein stain and immunoblotting with penta-His antibody. (B) His6-Vps4, His6-MIT (WT, I18D or L64D), and Ni-NTA beads were incubated with Bro1V. Bound material was visualized by both EZBiolab Instant-Band protein stain and immunoblotting with anti-Bro1 antiserum. (C) Vps4-specific activity with titration of Bro1V (10 nM to 8 µM). Vps4 (0.5 µM) ATPase assays were conducted using the indicated conditions and resolved by thin-layer chromatography for quantitation and calculation of hydrolysis rates. Dashed lines indicate Vps4-specific activity for 0.5 µM or 1.5 µM Vps4 alone, as indicated. Bro1V alone did not exhibit measurable ATP hydrolysis. (D) Vps4 titrations were performed with or without 4 µM Bro1V. Vps4-specific activity is presented. The vertical dotted line indicates the Vps4 apparent Km ±Bro1V. (E) V domains of S. cerevisiae Bro1, S. castellii Bro1, and H. sapiens HD-PTP (0.5–4 µM) were titrated against 0.5 µM S. cerevisiae Vps4. Specific activity of Vps4 is expressed as ADP generated per Vps4 molecule per minute. Data are represented as mean ± SEM.

V domain stimulates Vps4 ATPase activity in vitro. (A) GST-Bro1 fragments and GST-bound beads were incubated with His₆-Vps4. Bound material was visualized by both EZBiolab Instant-Band protein stain and immunoblotting with penta-His antibody. (B) His₆-Vps4, His₆-MIT (WT, I18D or L64D), and Ni-NTA beads were incubated with Bro1V. Bound material was visualized by both EZBiolab Instant-Band protein stain and immunoblotting with anti-Bro1 antiserum. (C) Vps4-specific activity with titration of Bro1V (10 nM to 8 µM). Vps4 (0.5 µM) ATPase assays were conducted using the indicated conditions and resolved by thin-layer chromatography for quantitation and calculation of hydrolysis rates. Dashed lines indicate Vps4-specific activity for 0.5 µM or 1.5 µM Vps4 alone, as indicated. Bro1V alone did not exhibit measurable ATP hydrolysis. (D) Vps4 titrations were performed with or without 4 µM Bro1V. Vps4-specific activity is presented. The vertical dotted line indicates the Vps4 apparent K_m ±Bro1V. (E) V domains of S. cerevisiae Bro1, S. castellii Bro1, and H. sapiens HD-PTP (0.5–4 µM) were titrated against 0.5 µM S. cerevisiae Vps4. Specific activity of Vps4 is expressed as ADP generated per Vps4 molecule per minute. Data are represented as mean ± SEM.