FIG. 3.

Ras1 cysteines 203 and 204 are palmitoylated in vivo. The ras1 mutant strain was transformed with plasmids expressing GFP-RAS1, GFP-ras1(C203S), GFP-ras1(C204S), and GFP-ras1(C203S, C204S) alleles under the control of the histone H3 promoter. Cell lysates from representative transformants were accessed for palmitoylation using the acyl-biotinyl switch assay. Each lysate was split between a hydroxylamine-positive (+) sample, a hydroxylamine-negative (−) sample, and a loading control sample. Proteins in the hydroxylamine-positive (+) and -negative (−) samples were labeled with biotin and precipitated using NeutrAvidin beads. Biotinylation was specific to proteins that contained a free sulfhydryl group generated by hydroxylamine cleavage of a thioester bond. Gfp-Ras1 proteins in the control samples were immunoprecipitated with anti-GFP antibodies. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride, and immunoblotted with anti-GFP antibodies. Biotinylated Gfp-Ras1 was detected in the GFP-RAS1, GFP-ras1(C203S), and GFP-ras1(C204S) lysates that included hydroxylamine, indicating that each of these lysates contained a Gfp-Ras1 protein with a thioester bond (i.e., palmitoylated).