Figure 6.

Purified Beclin 1 directly sensitizes the Ins(1,4,5)P₃R toward Ins(1,4,5)P₃, independently of Bcl-2. (A) Unidirectional ⁴⁵Ca²⁺-flux experiments in permeabilized HeLa cells with (gray; Beclin 1) or without (black; Ctrl) the addition of 2.5 µM purified Beclin 1. Mean fractional ⁴⁵Ca²⁺ release (%/2 min) as a function of time is shown in the absence or presence of 2.5 µM purified Beclin 1, added for 4 min, starting 2 min prior to the addition of 0.7 (circles) or 30 µM Ins(1,4,5)P₃ (triangles). The arrow indicates the addition of Ins(1,4,5)P₃. (B) Quantification of the ⁴⁵Ca²⁺ release triggered by the indicated concentration of Ins(1,4,5)P₃, normalized to control conditions (n = 4). (C) Mean traces of unidirectional ⁴⁵Ca²⁺-flux experiments in permeabilized HeLa cells without (black; Ctrl) or with 10 µM purified Beclin 1 domains (N-BH3: red; CCD: green; ECD-C: blue), added for 4 min, starting 2 min prior to the addition of 0.7 µM Ins(1,4,5)P₃. The arrow indicates the addition of Ins(1,4,5)P₃. (D) Quantification of the ⁴⁵Ca²⁺ release triggered by 0.7 µM Ins(1,4,5)P₃, normalized to control conditions, after addition of the indicated Beclin 1 domains (n = 4). (E) Representative example of pull-down experiments with GST-fused domains of Ins(1,4,5)P₃R1 and purified Beclin 1-F123A. The GST-fusion proteins were visualized using SYPRO Orange, while Beclin 1 was detected using anti-Beclin 1. IN = input. The asterisks indicate the different domains: ligand-binding domain (1–604); suppressor domain (1–225); Ins(1,4,5)P₃-binding core (226–604). (F) Representative unidirectional ⁴⁵Ca²⁺-flux experiment in permeabilized cells with (gray; Beclin 1-F123A) or without (black; Ctrl) 2.5 µM purified Beclin 1-F123A added for 4 min, starting 2 min prior to the addition of 0.7 µM Ins(1,4,5)P₃ (circles). The arrow indicates the addition of Ins(1,4,5)P₃. (G) Representative co-immunoprecipitation experiment of Beclin 1-F123A with Ins(1,4,5)P₃R1 from lysates of HeLa cells transfected with Beclin 1-F123A and pretreated with (3 h) or without (0 h) HBSS. IgG = negative control; IN = Input. (H) Representative co-immunoprecipitation experiment of Beclin 1 with Bcl-2 from lysates of HeLa cells transfected with Beclin 1 and pretreated with (3 or 5 h) or without (0 h) HBSS. IgG = negative control; IN = Input. *p < 0.05; **p < 0.01; N.S. not significant (paired t-test).