Fig. 5. R-Ras constructs and R-Ras palmitoylation. (A) GFP constructs used were wild-type R-Ras (pEGFP-R-Raswt), constitutively active R-Ras38V (pEGFP-R-Ras38V), dominant-negative R-Ras43 (pEGFP-R-Ras43N), constitutively active R-Ras deleted of the first 28 amino acids (pEGFP-R-Ras38V-NT), constitutively active R-Ras38V deleted of its last six amino acids (pEGFP-R-Ras38VdC), constitutively active R-Ras38V deleted of its HVR (175-212) but containing the last six amino acids (212-218) (pEGFP-R-Ras38V-dHVR), constitutively active R-Ras38V-2PA with two proline substituted on P202A and P203A (pEGFP-RRas38V-2PA), constitutively active R-Ras38V with a mutated palmitoylation site C213A (pEGFP-R-Ras38V-CA), constitutively active R-Ras38V with both palmitoylation and nucleotide destabilizing mutations C213A/S172P (pEGFP-R-Ras38V-SP) and the hypervariable region encoding 191-218aa of R-Ras (pEGFPHVR). (B) HeLa cells were transfected overnight with the following constructs: pEGFP-H-Ras61L (1), pEGFP-R-Ras38V-CA (2) and pEGFP-R-Ras38V (3). The cells were then labelled with [³H]palmitic acid for 4 hours, immunoprecipitated and analysed by SDS-PAGE. Note that the construct encoding EGFP-R-Ras with a mutated palmitoylation site (C213A; lane 2) is not labelled with palmitate, whereas EGFP-H-Ras61L (lane 1) is strongly labelled, and EGFP-R-Ras38V (lane 3) to a lesser extent.