Consequences of altered isoprenylation targets ona-factor export and bioactivity.

Cysteine-containing amino acid sequences (CAAX,CC, and CXC; C is cysteine, A is any aliphatic amino acid, and X is any aminoacid) are targets for the attachment of C15 (farnesyl) and C20 (geranylgeranyl)isoprenoids to peptides and proteins by specific prenyltransferases. Althoughmuch work has centered on the enzymatic mechanisms of these enzymes, thebiological consequences of the differential isoprenylation they catalyze remainto be elucidated. Farnesylation of the a-factor mating pheromone ofSaccharomyces cerevisiae is a known prerequisite for its biological activity andits secretion through a pathway utilizing the yeast STE6 protein, a homolog ofthe mammalian multidrug resistance (MDR) P-glycoprotein. We generated specificmutations in the a-factor gene to encode isoprenylation targets forgeranylgeranylation [Cys-Val-Ile-Leu (CVIL) and Ser-Val-Cys-Cys (SVCC)] in placeof the natural farnesylation motif [Cys-Val-Ile-Ala (CVIA)]. The a-factorscontaining these modified prenylation sites were successfully exported by aSTE6-dependent mechanism. Furthermore, these peptides, as well as syntheticgeranylgeranyl a-factor, retained bioactivity. Chromatographic comparisons ofsynthetic and biosynthetic pheromones suggest that, in vivo, a peptide substratecontaining the geranylgeranylation target CVIL can be both farnesylated andgeranylgeranylated. These results clearly demonstrate that in vivo (i) differentprenyltransferases may recognize the same substrate; (ii) both farnesylated andgeranylgeranylated a-factor peptides are substrates for export via STE6, aMDR-like protein; and (iii) farnesylated and geranylgeranylated pheromones areboth biologically active.