Figure 1.

Prenylcysteine carboxy methyltransferase activity in Dictyostelium cells and cell-free extracts. (A) AFC is an inhibitor of protein carboxy methylation. AFC inhibition of protein carboxy methylation is localized to the 20- to 25-kDa region of a denaturing gel. (B) AFC inhibits GTPγS stimulation. GTPγS can increase the level of methylation by >300%, and AFC can inhibit this increase by >50%. In experiments A and B, Dictyostelium cellular extracts were labeled with [³H]SAM in the presence of AFC, GTPγS, or both. (C) Transient increase of prenylcysteine carboxy methylation after cAMP stimulation. Cells starved for 1 h were pulsed with cAMP for 5 h and labeled for 1 h with [³H-methyl]methionine in vivo. cAMP was then added to 1 μM. The reactions were stopped at the indicated intervals, and proteins were separated by SDS-PAGE. Protein levels were determined as described in Materials and Methods. (D) Aliquots containing 150 μg of protein from extracts of wild-type AX4 or icmA deletion (KO) cells overexpressing TAP-tagged YFP-RasG were incubated with SAM. Inset, TAP-YFP-RasG region of the gel, 68 kDa. The inset shows fractions 1–8 at a different scale, demonstrating that this region is methylated in AX4 but not in the mutant.