ABHD17A has at least four stable acylation sites that are required for plasma membrane localization. (A) An APE assay detects at least four S-acylation sites in ABHD17A. The addition of a mass tag causes a size shift that is proportional to the number of acyl modifications; the number of acylation sites is denoted by asterisks. The doubly acylated calnexin serves as a control. (B and C) The first 17 residues of ABHD17A localize to the plasma membrane. The white line through the cell was used for the line scan in C. (D and E) The plasma membrane localization of the first 17 residues of ABHD17A is lost when cysteine residues are mutated to serine. A line scan (E) depicts the higher signal intensity in the cytosol of the cell when the N-terminal cysteine residues are mutated to serine of ABHD17A. (B and D) show representative confocal images of fixed cells, with the intensity increased to show the plasma membrane localization of WT compared to the cytosolic signal of 4C>S. (F) ABHD17A acylation does not show significant turnover as measured by 17-ODYA labeling followed by a chase in the presence or absence of 10 µM HDFP. (G) Quantification of ABHD17A acylation in F. Error bars indicate standard deviation (STDEV). (H) NRas acylation does show significant turnover when measured with click-labeling followed by a chase in the presence or absence of 10 µM HDFP, as described in F. (I) Quantification of NRas acylation in H. Source data are available for this figure: SourceData FS1.