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. 2022 Jan 19;298(3):101600. doi: 10.1016/j.jbc.2022.101600

Figure 8.

Figure 8

The interaction of PSP94 with CRISP2 is sensitive to calcium. A and B, CRISP2 and PSP94 both bind calcium in vitro and the interaction between CRISP2 and PSP94 is disrupted at high calcium concentrations. Calcium binding by CRISP2 and PSP94 was assessed by MST (A). Formation of the heteromeric complex between CRISP2 and PSP94 in the presence of 10 mM CaCl2 (B) was assessed by microscale thermophoresis. Measurements were performed in triplicates, and the corresponding dissociation constants (Kd) are indicated. C, the interaction between CRISP2 and PSP94 is disrupted by calcium in vivo. Cells expressing FLAG-tagged PSP94 alone (a) or together with an HA-tagged CRISP2 (b) were cultivated in media containing the indicated concentration of CaCl2 (0, 5, 10 mM). The interaction between PSP94 and CRISP2 in the culture medium was analyzed by immunoprecipitation with an anti-HA antibody followed by Western blotting to detect both HA-tagged CRISP2 and FLAG-tagged PSP94. D, quantification of the interaction between CRISP2 and PSP94. Interaction between these two proteins detected by coimmunoprecipitation was quantified and plotted as ratio between the signal obtained from cells expressing both proteins, CRISP2-HA and PSP94-FLAG (b), and the background signal from cells lacking CRISP2-HA (a). Data represent mean ± SD of three independent determinations, and statistical significance is indicated: ∗p ≤ 0.05. E, calcium releases the inhibitory action of PSP94 on sterol export by CRISP2. Quadruple mutant cells (pry1Δ pry2Δ say1Δ hem1Δ) expressing either CRISP2 alone or together with PSP94 were cultivated in media containing the indicated concentrations of CaCl2 (0, 5, 10 mM) and labeled with [14C]cholesterol. Lipids were extracted from the cell pellet (P) and the culture supernatant (S), separated by TLC, and quantified. F, export of radiolabeled cholesteryl acetate is plotted as export index in the graph. Data correspond to means ± SD of three independent determinations, and statistical significance is indicated: ∗∗p ≤ 0.01. N/A, not applicable.