Ub potentiates V domain stimulation of Vps4 ATPase activity. (A) Vps4 (0.5 µM) ATPase specific activity in the presence of 1 µM S. cerevisiae Bro1V, S. cerevisiae Bro1VΔUBD (I377R), S. castellii Bro1V (370–708), and S. castellii Bro1VΔUBD (I377R) ± 50 µM mono-Ub. Data are represented as mean ± SEM. Asterisks indicate a statistically significant difference compared with Vps4 alone (P < 0.05), and number signs indicate a statistically significant difference compared with Vps4 + Bro1V − Ub (P < 0.05). (B) Bro1V titration performed in the presence of 0.5 µM Vps4 with or without 50 µM Ub. The vertical dotted line indicates the Bro1V concentration generating half-maximal stimulation within each context. (C) Vps4 titration (0.05–1.0 µM) in the presence of 4 µM Bro1V WT or Ub-binding mutant (L386R) and 50 µM Ub. Vps4-specific activity (ADP generated per Vps4 molecule per minute) is presented. Data are represented as mean ± SEM. Vertical dotted lines indicate the Vps4 apparent Km in each context. (D) The Vps4 apparent Km of Vps4 alone, Vps4 + 4 µM Bro1V, Vps4 + 4 µM Bro1V + 50 µM Ub, Vps4 + 4 µM Bro1VΔUBD (L386R), and Vps4 + 4 µM Bro1VΔUBD + 50 µM Ub was determined from Vps4 titration experiments in Figs. 4 D, 9 C, and S4 C. Asterisks indicate a statistically significant difference (P < 0.02). (E) Vps4 ATPase activity (0.5 µM) in the presence of 4 µM S. cerevisiae Bro1V, Bro1VM4, Bro1VM8, or Bro1VM10 without or with 50 µM Ub. Vps4-specific activity with Ub addition is normalized to activity without Ub. Data are represented as mean ± SEM. Asterisk indicates a statistically significant difference with or without Ub addition (P = 0.0012). (F) NBD-PC– and FM4-64–stained WT (SEY6210), bro1Δ (GOY65), and GOY65 reexpressing Bro1 or overexpressing Bro1ΔBOD, Bro1ΔBOD,ΔUBD (I377R, L386R), and Bro1ΔBOD,M8 were analyzed by live-cell fluorescence microscopy and quantified for the frequency of cells with NBD-PC in the vacuolar lumen. Data are represented as mean ± SEM. Asterisks indicate a statistically significant difference (P < 0.01), and number signs indicate a statistically significant difference compared with bro1Δ (P < 0.005).
Figure 9.

Ub potentiates V domain stimulation of Vps4 ATPase activity. (A) Vps4 (0.5 µM) ATPase specific activity in the presence of 1 µM S. cerevisiae Bro1V, S. cerevisiae Bro1VΔUBD (I377R), S. castellii Bro1V (370–708), and S. castellii Bro1VΔUBD (I377R) ± 50 µM mono-Ub. Data are represented as mean ± SEM. Asterisks indicate a statistically significant difference compared with Vps4 alone (P < 0.05), and number signs indicate a statistically significant difference compared with Vps4 + Bro1V − Ub (P < 0.05). (B) Bro1V titration performed in the presence of 0.5 µM Vps4 with or without 50 µM Ub. The vertical dotted line indicates the Bro1V concentration generating half-maximal stimulation within each context. (C) Vps4 titration (0.05–1.0 µM) in the presence of 4 µM Bro1V WT or Ub-binding mutant (L386R) and 50 µM Ub. Vps4-specific activity (ADP generated per Vps4 molecule per minute) is presented. Data are represented as mean ± SEM. Vertical dotted lines indicate the Vps4 apparent Km in each context. (D) The Vps4 apparent Km of Vps4 alone, Vps4 + 4 µM Bro1V, Vps4 + 4 µM Bro1V + 50 µM Ub, Vps4 + 4 µM Bro1VΔUBD (L386R), and Vps4 + 4 µM Bro1VΔUBD + 50 µM Ub was determined from Vps4 titration experiments in Figs. 4 D, 9 C, and S4 C. Asterisks indicate a statistically significant difference (P < 0.02). (E) Vps4 ATPase activity (0.5 µM) in the presence of 4 µM S. cerevisiae Bro1V, Bro1VM4, Bro1VM8, or Bro1VM10 without or with 50 µM Ub. Vps4-specific activity with Ub addition is normalized to activity without Ub. Data are represented as mean ± SEM. Asterisk indicates a statistically significant difference with or without Ub addition (P = 0.0012). (F) NBD-PC– and FM4-64–stained WT (SEY6210), bro1Δ (GOY65), and GOY65 reexpressing Bro1 or overexpressing Bro1ΔBOD, Bro1ΔBOD,ΔUBD (I377R, L386R), and Bro1ΔBOD,M8 were analyzed by live-cell fluorescence microscopy and quantified for the frequency of cells with NBD-PC in the vacuolar lumen. Data are represented as mean ± SEM. Asterisks indicate a statistically significant difference (P < 0.01), and number signs indicate a statistically significant difference compared with bro1Δ (P < 0.005).

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