Figure 2.
A targeting-independent role for the ABHD17A N-terminus. (A) Specific cysteine combinations are required for the activity of FLAG-tagged ABHD17A on NRas. Acylation of GFP-NRas was detected by click-labeling with 17-ODYA (upper panel). Levels of immunoprecipitated NRas (IP; middle panel), or total ABHD17A (lysate; lower panel), were detected by western blot using anti-GFP or FLAG antibody, respectively. (B) Quantification of GFP-NRas acylation is shown in A. NRas acylation was normalized to vector. One-way ANOVA with Tukey’s multiple comparison test; n = 3, ns = P > 0.05, * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001. Statistical analysis is depicted with black font (comparison to vector) and blue font (comparison to WT). Error bars indicate STDEV. (C) Schematic of ABHD17A constructs. (D) The Src myristoylation motif restored plasma membrane localization of ABHD17A lacking either the first 19 residues, or N-terminal cysteine residues, as quantified by BRET analysis. One-way ANOVA with Tukey’s multiple comparison test; n = 5, ns = P > 0.05, ** = P ≤ 0.01. Error bars indicate STDEV. (E) Recruiting non-acylated ABHD17A to the plasma membrane with a Src motif did not restore activity on NRas. Acylation of GFP-NRas was detected by click-labeling with 17-ODYA (upper panel). Levels of immunoprecipitated NRas (middle panel), or total ABHD17A (lower panel), were detected by western blot using anti GFP or FLAG antibody, respectively. (F) Quantification of GFP-NRas acylation in E. NRas acylation was normalized to vector. One-way ANOVA with Tukey’s multiple comparison test; n = 3, ns = P > 0.05, *** = P ≤ 0.001, **** = P ≤ 0.0001. Statistical analysis is depicted with black font (comparison to vector) and blue font (comparison to WT). Error bars indicate STDEV. (G) Schematic of ABHD17A constructs with hydrophobic residues in place of N-terminal cysteine residues. (H) N-terminal hydrophobic residues localize ABHD17A to the plasma membrane when paired with the Src motif as quantified by BRET analysis. One-way ANOVA with Tukey’s multiple comparison test; n = 5, ns = P > 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001. Error bars indicate STDEV. (I) Pairing the Src motif with hydrophobic N-terminal residues restored activity on GFP-NRas. Acylation of GFP-NRas was detected by click-labeling with 17-ODYA (upper panel). Levels of immunoprecipitated NRas (middle panel), or total ABHD17A (lower panel), were detected by western blot using anti GFP or FLAG antibody, respectively. (J) Quantification of NRas acylation in I. NRas acylation was normalized to vector. One-way ANOVA with Tukey’s multiple comparison test; n = 3, ns = P > 0.05, ** = P ≤ 0.01. Statistical analysis is depicted with black font (comparison to vector) and blue font (comparison to WT). Error bars indicate STDEV. Source data are available for this figure: SourceData F2. Refer to the image caption for details.

A targeting-independent role for the ABHD17A N-terminus. (A) Specific cysteine combinations are required for the activity of FLAG-tagged ABHD17A on NRas. Acylation of GFP-NRas was detected by click-labeling with 17-ODYA (upper panel). Levels of immunoprecipitated NRas (IP; middle panel), or total ABHD17A (lysate; lower panel), were detected by western blot using anti-GFP or FLAG antibody, respectively. (B) Quantification of GFP-NRas acylation is shown in A. NRas acylation was normalized to vector. One-way ANOVA with Tukey’s multiple comparison test; n = 3, ns = P > 0.05, * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001. Statistical analysis is depicted with black font (comparison to vector) and blue font (comparison to WT). Error bars indicate STDEV. (C) Schematic of ABHD17A constructs. (D) The Src myristoylation motif restored plasma membrane localization of ABHD17A lacking either the first 19 residues, or N-terminal cysteine residues, as quantified by BRET analysis. One-way ANOVA with Tukey’s multiple comparison test; n = 5, ns = P > 0.05, ** = P ≤ 0.01. Error bars indicate STDEV. (E) Recruiting non-acylated ABHD17A to the plasma membrane with a Src motif did not restore activity on NRas. Acylation of GFP-NRas was detected by click-labeling with 17-ODYA (upper panel). Levels of immunoprecipitated NRas (middle panel), or total ABHD17A (lower panel), were detected by western blot using anti GFP or FLAG antibody, respectively. (F) Quantification of GFP-NRas acylation in E. NRas acylation was normalized to vector. One-way ANOVA with Tukey’s multiple comparison test; n = 3, ns = P > 0.05, *** = P ≤ 0.001, **** = P ≤ 0.0001. Statistical analysis is depicted with black font (comparison to vector) and blue font (comparison to WT). Error bars indicate STDEV. (G) Schematic of ABHD17A constructs with hydrophobic residues in place of N-terminal cysteine residues. (H) N-terminal hydrophobic residues localize ABHD17A to the plasma membrane when paired with the Src motif as quantified by BRET analysis. One-way ANOVA with Tukey’s multiple comparison test; n = 5, ns = P > 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001. Error bars indicate STDEV. (I) Pairing the Src motif with hydrophobic N-terminal residues restored activity on GFP-NRas. Acylation of GFP-NRas was detected by click-labeling with 17-ODYA (upper panel). Levels of immunoprecipitated NRas (middle panel), or total ABHD17A (lower panel), were detected by western blot using anti GFP or FLAG antibody, respectively. (J) Quantification of NRas acylation in I. NRas acylation was normalized to vector. One-way ANOVA with Tukey’s multiple comparison test; n = 3, ns = P > 0.05, ** = P ≤ 0.01. Statistical analysis is depicted with black font (comparison to vector) and blue font (comparison to WT). Error bars indicate STDEV. Source data are available for this figure: SourceData F2.

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