Palmitoylation targets the S0-S1 linker to the plasma membrane. A schematic of the short S0-S1 linker YFP fusion construct (S0-S1-YFP), that encodes the 70 amino acid intracellular S0-S1 linker between amino acids Arg-44 and Arg-11 fused in-frame with YFP, and relative position of the palmitoylated cysteine residues. A, representative single cell confocal images of the S0-S1-YFP linker, S0-S1 C53:54:56A-YFP linker, and the S0-S1-YFP linker fusion proteins after incubation with the palmitoylation inhibitor 2BP (100 μm) for 24 h, expressed in HEK293 cells (scale bars: 10 μm). B, summary bar graph illustrates the effect of site-directed mutagenesis of cysteine residues in the S0-S1 linker on localization of the respective S0-S1-YFP fusion protein at the plasma membrane expressed as a percentage of the wild-type S0-S1-YFP fusion protein (where wild-type S0-S1-YFP membrane expression is normalized to 100%). (For all S0-S1 linker constructs, N>3, n>330 cells analyzed). **, p < 0.01; ***, p < 0.001 compared with wild-type S0-S1 (ANOVA with Tukey post hoc test).