Fig. 1. Illustration of the linker-GFP construct with the location of the consensus myristoylation sequence and the positions where the mutations were introduced (A), expression of the recombinant proteins in COS-7 cells (B) and fatty acylation of the GFP chimeras (C). By recursive PCR, the triplet AGS repeated nine times was created following a consensus N-myristoylation sequence and fused in frame to the GFP sequence. Mutations were introduced at Gly2, Cys3, Ser9, Ser15 and Ser21 in various combinations, as described in the Materials and Methods (A). The different linker-GFP constructs were inserted in a pCDNA3 vector that was used to transfect COS-7 cells (B). Transfected COS-7 cells were starved for 1 hour in DMEM without serum and were then metabolically labeled for 4 hours with either [³H]-myristic acid (Myr) or [³H]-palmitic acid (Palm). Cell lysates were immunoprecipitated with an anti-GFP antibody, analyzed by SDS-PAGE and exposed to a film as described in the Materials and Methods. Identical results were obtained in two independent metabolic labeling experiments.

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