Fig. 4.

Cys-195 of Ykt6 is farnesylated in vivo.(A) HeLa cells were treated for 2 days with 30 μM FTI-277 or 20 μM GGTI-298, fixed, and coimmunostained with anti-ykt6 and anti-p115 antibodies. (Bar = 10 μm.) (B) HeLa cells were treated for 1 day with 30 μM lovastatin or for 2 days with 30 μM FTI-277 or 20 μM GGTI-298. Cells were solubilized with 2% Triton X-114 on ice and partitioned into aqueous (A) and detergent (D) phases at 37°C. Ykt6 derived from both phases was analyzed by immunoblot with anti-ykt6 antibodies. (C) HeLa cells were transfected with GFP constructs fused with the wild-type Ykt6 or Ykt6-F42E mutant. After a 15-h incubation with [³H]mevalonolactone, both forms of GFP-ykt6 containing the single or double substitutions of the carboxyl-terminal cysteines were immunoprecipitated from cell lysates with antibodies to GFP. Each immunoprecipitate was resolved by SDS/PAGE and subjected to fluorography to visualize prenylated proteins (a) or to immunoblot analysis using anti-GFP antibody (b).