Figure 2. Phospho mutants of the acidic... | Rockefeller University Press

Figure 2.

$Phospho mutants of the acidic tract alter the ACBD5–VAPB interaction and its phosphatase sensitivity.(A) Schematic overview of ACBD5 domain structure; amino acid sequences of the phosphorylation sites mutated in this study in bold. (B) Schematic model of the interaction between the ACBD5 FFAT-like motif and the VAPB MSP domain. The interaction occurs in two steps (Furuita et al., 2010): (1) Initial nonspecific electrostatic interaction between the FFAT acidic tract and the basic electropositive surface of the MSP domain; (2) specific binding of the FFAT core to the FFAT-binding site of the MSP domain, which consists of an electropositive face. (C–E) ACBD5 constructs with nonphosphorylatable (A) and phosphomimetic (E) residues in the acidic tract were expressed in COS-7 cells. The proteins were immunoprecipitated, and endogenous bound VAPB was detected by immunoblotting using Myc/VAPB antibodies. In C, data were analyzed by one-way ANOVA with Dunnett’s multiple comparison test. Total VAPB (IP fraction) was normalized against total VAPB (input) and Myc-ACBD5 (IP fraction). In D and E, lysates were treated ± λPP before IP. Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison test (D) or a two-tailed unpaired t test (E). Total VAPB (IP fraction) was normalized against Myc-ACBD5 (IP fraction). VAPB signal in the treated sample was then calculated as a percentage of VAPB signal in the untreated sample. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. Results of at least three independent IPs were quantified. Source data are available for this figure: SourceData F2.$

Phospho mutants of the acidic tract alter the ACBD5–VAPB interaction and its phosphatase sensitivity. (A) Schematic overview of ACBD5 domain structure; amino acid sequences of the phosphorylation sites mutated in this study in bold. (B) Schematic model of the interaction between the ACBD5 FFAT-like motif and the VAPB MSP domain. The interaction occurs in two steps (Furuita et al., 2010): (1) Initial nonspecific electrostatic interaction between the FFAT acidic tract and the basic electropositive surface of the MSP domain; (2) specific binding of the FFAT core to the FFAT-binding site of the MSP domain, which consists of an electropositive face. (C–E) ACBD5 constructs with nonphosphorylatable (A) and phosphomimetic (E) residues in the acidic tract were expressed in COS-7 cells. The proteins were immunoprecipitated, and endogenous bound VAPB was detected by immunoblotting using Myc/VAPB antibodies. In C, data were analyzed by one-way ANOVA with Dunnett’s multiple comparison test. Total VAPB (IP fraction) was normalized against total VAPB (input) and Myc-ACBD5 (IP fraction). In D and E, lysates were treated ± λPP before IP. Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison test (D) or a two-tailed unpaired t test (E). Total VAPB (IP fraction) was normalized against Myc-ACBD5 (IP fraction). VAPB signal in the treated sample was then calculated as a percentage of VAPB signal in the untreated sample. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. Results of at least three independent IPs were quantified. Source data are available for this figure: SourceData F2.

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