Significance of C-terminal cysteine modifications to the biological activity of the Saccharomyces cerevisiae a-factor mating pheromone.

Abstract

We have undertaken total synthesis of the Saccharomyces cerevisiae a-factor (NH2-YIIKGVFWDPAC[Sfarnesyl]-COOCH3) and several Cys-12 analogs to determine the significance of S-farnesylation and carboxyterminal methyl esterification to the biological activity of this lipopeptide mating pheromone. Replacement of either the farnesyl group or the carboxy-terminal methyl ester by a hydrogen atom resulted in marked reduction but not total loss of bioactivity as measured by a variety of assays. Moreover, both the farnesyl and methyl ester groups could be replaced by other substituents to produce biologically active analogs. The bioactivity of a-factor decreased as the number of prenyl units on the cysteine sulfur decreased from three to one, and an a-factor analog having the S-farnesyl group replaced by an S-hexadecanyl group was more active than an S-methyl a-factor analog. Thus, with two types of modifications, a-factor activity increased as the Salkyl group became bulkier and more hydrophobic. MATa cells having deletions of the a-factor structural genes (mfal mfa2 mutants) were capable of mating with either sst2 or wild-type MATa cells in the presence of exogenous a-factor, indicating that it is not absolutely essential for MATa cells to actively produce a-factor in order to mate. Various a-factor analogs were found to partially restore mating to these strains as well, and their relative activities in the mating restoration assay were similar to their activities in the other assays used in this study. Mating was not restored by addition of exogenous a-factor to a cross of a wild-type MATa strain and a MATaste6 mutant, indicating a role of the STE6 gene product in mating in addition to its secretion of a-factor.

The Yeast a-factor Transporter Ste6p, a Member of the ABC Superfamily, Couples ATP Hydrolysis to Pheromone Export

“…These values compare well with the nanomolar range required to elicit a biological response (8). Equally or perhaps more importantly, these values are also on par with the concentration of a-factor observed in the culture media of actively growing MATa cells (53). For this reason, we suggest that Ste6p possesses an inherently low ATPase activity that is necessary for the transport mechanism and sufficient to generate physiological concentrations of extracellular a-factor.…”

Biogenesis of the Saccharomyces cerevisiae Pheromone a -Factor, from Yeast Mating to Human Disease

“…Numerous mutational alterations of residues within the mature portion of a-factor prevent the activation of Ste3, without affecting the biogenesis or export of a-factor (44,116). Synthetic versions of a-factor lacking the methyl group, the farnesyl group, or both are significantly defective in the mating and halo assays discussed below, possessing 1%, 0.1%, and 0.0025%, respectively, of the activity level of fully CAAX-modified a-factor (41,172). Small alterations of the farnesyl moiety can also influence a-factor activity (74).…”

“…3). In addition, the prenyl group appears to be directly required for the recognition of a-factor by its processing enzymes (41,44,112,172,231). Interestingly, when a-factor is forced to undergo geranylgeranylation instead of farnesylation (by mutation of its CAAX motif from CVIA to CVIL), there is no impact on a-factor processing, export, or mating, indicating that either form of prenylation is sufficient for maintaining the biological properties of a-factor (43).…”

“…These include genetic studies to identify mating-defective sterile (ste) mutants (1,94,166,167,270,271), biochemical analyses to determine the activity corresponding to the mutant gene (7, 18, 33, 108, 112-114, 136, 186, 233, 237, 255), pulse-chase metabolic labeling experiments to determine which step of a-factor biogenesis is blocked in mutant cells (1,26,56,94,116,150,231), and mutational analysis of MFA1 to define the residues of a-factor required for recognition by the biogenesis enzymes (43,44,56,116,172,260). Most commonly, SDS-PAGE has been used to examine the 35 S-labeled a-factor biosynthetic intermediates present in intracellular and extracellular fractions from wild-type and mutant cells (Fig.…”

Mammalian Prenylcysteine Carboxyl Methyltransferase Is in the Endoplasmic Reticulum

“…Although processed CAAX proteins can associate with phospholipid vesicles in vitro (4), it is not known whether membrane proteins participate in prenylcysteine membrane association in vivo. The Saccharomyces cerevisiae mating pheromone, a-factor, is a CAAX-processed polypeptide, and both its secretion via the Ste6p transporter (5) and its engagement of the Ste3p G protein-linked receptor (6) are dependent on prenylcysteine carboxyl methylation, suggesting a role for this modification in protein-protein interactions. A cycle of prenylcysteine carboxyl methylation is associated with neutrophil activation (7), and inhibitors of this enzyme block signal transduction in neutrophils (7), macrophages (8), and platelets (9), suggesting that, like bacterial chemotaxis (10), some eukaryotic processes may be regulated by reversible carboxyl methylation.…”