Analysis of protein prenylation and carboxyl-methylation

This unit describes methods for analysis of prenylation and the carboxyl-methylation that often accompanies it. The two prenoid groups that have been found attached to proteins--farnesyl (C15) and geranylgeranyl (C20)--are both derived from intermediates in the isoprenoid biosynthetic pathway that utilizes mevalonic acid. In the protocols described here, radiolabeled mevalonate is used to label these intermediates in either intact cells or in vitro; the labeled intermediates then become incorporated into proteins. Alternatively, the preformed radioactive prenyl diphosphates can be used for in vitro translations, as described here. Carboxyl-methylation of C-terminal prenylated cysteine residues often occurs subsequent to prenylation. Methods are given for radiolabeling of the methyl group with [(3)H-methyl]methionine, that is converted intracellularly into S-adenosylmethionine, and for radiolabeling with preformed S-adenosyl[(3)H]methionine.