Figure 1.
The N-terminus is necessary and sufficient for S-acylation and plasma membrane localization. (A) Plasma membrane localization of WT ABHD17A measured with BRET is lost with either mutation of N-terminal cysteine residues or deletion of the N-terminus. Cells were transiently transfected to co-express Rluc8-tagged ABHD17A mutants with a Venus-tagged organelle marker for the plasma membrane, Golgi, or ER. One-way ANOVA with Tukey’s multiple comparison test; n = 5, ns = P > 0.05, ** = P ≤ 0.01, **** = P ≤ 0.0001. Error bars depict STDEV. (B) AF2-predicted structure of ABHD17A showing the first 19 residues in blue. (C) Sequence of the first 19 residues of ABHD17A showing the putative acylated cysteine residues in blue. (D) The ABHD17A N-terminus contains a predicted α-helix. Cysteine residues are shown in blue with acyl groups indicated by blue wavy lines attached to Cys10, 11, 14, and 15. (E) Acylation of ABHD17A is abolished when either the first four or five N-terminal cysteine residues are mutated to serine as visualized with a click-labeling assay using the palmitate analog 17-ODYA. Upper panel shows 17-ODYA signal and lower panel shows a western blot of total protein detected with FLAG antibody. (F) The first 17 residues of ABHD17A (ABHD17A1–17) are sufficient for acylation. 17-ODYA signal was detected by click-labeling (upper panel); total protein was detected with GFP antibody (lower panel). (G) Plasma membrane localization of the first 17 residues of ABHD17A requires N-terminal cysteine residues. The BRET assay is described in A. One-way ANOVA with Tukey’s multiple comparison test; n = 5, ns = P > 0.05, **** = P ≤ 0.0001. Error bars indicate STDEV. (H) ABHD17A-FLAG acylation in double cysteine mutants. Acylation of ABHD17A-FLAG was detected by click-labeling with 17-ODYA (upper panel). Levels of immunoprecipitated ABHD17A were detected by western blot using anti FLAG antibody (lower panel). (I) Quantification ABHD17A-FLAG acylation in H. One-way ANOVA with Tukey’s multiple comparison test; n = 3, ns = P > 0.05, * = P ≤ 0.05, ** = P ≤ 0.01, **** = P ≤ 0.0001. Statistical analysis shows comparison to WT ABHD17A. Error bars indicate STDEV. Source data are available for this figure: SourceData F1. Refer to the image caption for details.

The N-terminus is necessary and sufficient for S-acylation and plasma membrane localization. (A) Plasma membrane localization of WT ABHD17A measured with BRET is lost with either mutation of N-terminal cysteine residues or deletion of the N-terminus. Cells were transiently transfected to co-express Rluc8-tagged ABHD17A mutants with a Venus-tagged organelle marker for the plasma membrane, Golgi, or ER. One-way ANOVA with Tukey’s multiple comparison test; n = 5, ns = P > 0.05, ** = P ≤ 0.01, **** = P ≤ 0.0001. Error bars depict STDEV. (B) AF2-predicted structure of ABHD17A showing the first 19 residues in blue. (C) Sequence of the first 19 residues of ABHD17A showing the putative acylated cysteine residues in blue. (D) The ABHD17A N-terminus contains a predicted α-helix. Cysteine residues are shown in blue with acyl groups indicated by blue wavy lines attached to Cys10, 11, 14, and 15. (E) Acylation of ABHD17A is abolished when either the first four or five N-terminal cysteine residues are mutated to serine as visualized with a click-labeling assay using the palmitate analog 17-ODYA. Upper panel shows 17-ODYA signal and lower panel shows a western blot of total protein detected with FLAG antibody. (F) The first 17 residues of ABHD17A (ABHD17A1–17) are sufficient for acylation. 17-ODYA signal was detected by click-labeling (upper panel); total protein was detected with GFP antibody (lower panel). (G) Plasma membrane localization of the first 17 residues of ABHD17A requires N-terminal cysteine residues. The BRET assay is described in A. One-way ANOVA with Tukey’s multiple comparison test; n = 5, ns = P > 0.05, **** = P ≤ 0.0001. Error bars indicate STDEV. (H) ABHD17A-FLAG acylation in double cysteine mutants. Acylation of ABHD17A-FLAG was detected by click-labeling with 17-ODYA (upper panel). Levels of immunoprecipitated ABHD17A were detected by western blot using anti FLAG antibody (lower panel). (I) Quantification ABHD17A-FLAG acylation in H. One-way ANOVA with Tukey’s multiple comparison test; n = 3, ns = P > 0.05, * = P ≤ 0.05, ** = P ≤ 0.01, **** = P ≤ 0.0001. Statistical analysis shows comparison to WT ABHD17A. Error bars indicate STDEV. Source data are available for this figure: SourceData F1.

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