Total in vitro maturation of the Saccharomyces cerevisiae a-factor lipopeptide mating pheromone.
The a-factor mating pheromone, produced by Saccharomyces cerevisiae a haploid cells, is post-translationally modified in a manner analogous to that of the ras proto-oncogene product. A consensus C-terminal amino acid sequence, -CAAX (C is cysteine, A is aliphatic amino acid, and X is any amino acid), is the target of these modifications, which include isoprenylation (essential for Ras function), proteolysis of the -AAX sequence, and carboxy methyl esterification. Recently, the RAM/DPR1 gene product was shown to be a component of the activity responsible for isoprenylation of both Ras and a-factor. In this report, we present an in vitro assay which not only detects a-factor isoprenylation, but also proteolysis and carboxy methyl esterification, and directly demonstrates, biochemically, the order of these processing events. This a-factor maturation assay may prove useful for screening agents which block any of the steps involved in the post-translational modification of the a-factor and Ras -CAAX sequences. Such agents would be potential anti-Ras-related cancer therapeutic drugs.
Marcus
S
S
Department of Microbiology, University of Tennessee, Knoxville 37996.
Caldwell
G A
GA
Xue
C B
CB
Naider
F
F
Becker
J M
JM
D000595:Amino Acid Sequence; D000970:Antineoplastic Agents; D001483:Base Sequence; D016384:Consensus Sequence; D000072235:Mating Factor; D008745:Methylation; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development*
D000970:Antineoplastic Agents; D010455:Peptides; D011518:Proto-Oncogene Proteins; D000072235:Mating Factor