Consequences of altered isoprenylation targets on a-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 amino acid) are targets for the attachment of C15 (farnesyl) and C20 (geranylgeranyl) isoprenoids to peptides and proteins by specific prenyltransferases. Although much work has centered on the enzymatic mechanisms of these enzymes, the biological consequences of the differential isoprenylation they catalyze remain to be elucidated. Farnesylation of the a-factor mating pheromone of Saccharomyces cerevisiae is a known prerequisite for its biological activity and its secretion through a pathway utilizing the yeast STE6 protein, a homolog of the mammalian multidrug resistance (MDR) P-glycoprotein. We generated specific mutations in the a-factor gene to encode isoprenylation targets for geranylgeranylation [Cys-Val-Ile-Leu (CVIL) and Ser-Val-Cys-Cys (SVCC)] in place of the natural farnesylation motif [Cys-Val-Ile-Ala (CVIA)]. The a-factors containing these modified prenylation sites were successfully exported by a STE6-dependent mechanism. Furthermore, these peptides, as well as synthetic geranylgeranyl a-factor, retained bioactivity. Chromatographic comparisons of synthetic and biosynthetic pheromones suggest that, in vivo, a peptide substrate containing the geranylgeranylation target CVIL can be both farnesylated and geranylgeranylated. These results clearly demonstrate that in vivo (i) different prenyltransferases may recognize the same substrate; (ii) both farnesylated and geranylgeranylated a-factor peptides are substrates for export via STE6, a MDR-like protein; and (iii) farnesylated and geranylgeranylated pheromones are both biologically active.
Caldwell
G A
GA
Department of Microbiology, University of Tennessee, Knoxville 37996.
Wang
S H
SH
Naider
F
F
Becker
J M
JM
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001692:Biological Transport; D004122:Dimethylallyltranstransferase / Q000378:metabolism; D004224:Diterpenes / Q000378:metabolism; D005204:Farnesol / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D006023:Glycoproteins; D000072235:Mating Factor; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009842:Oligopeptides / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D017368:Protein Prenylation; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D018528:ATP-Binding Cassette Transporters; D004224:Diterpenes; D005656:Fungal Proteins; D006023:Glycoproteins; D009842:Oligopeptides; D010455:Peptides; C489277:STE6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D005204:Farnesol; D000072235:Mating Factor; C017338:geranylgeraniol; D004122:Dimethylallyltranstransferase