Role of prenylation in the interaction of the a-factor mating pheromone with phospholipid bilayers

Abstract

We have studied the interaction between phospholipids and a-factor (YIIKGVFWDPAC-[Farn]OMe), S-alkylated forms of a-factor with the farnesyl group substituted by methyl, hexadecanyl, or benzyl groups, and truncated forms of this lipopeptide. Circular dichroism studies suggest that, despite its lack of farnesylation, S-methyl-a-factor is incorporated into vesicles of dimyristoylphosphatidylcholine in a conformation similar to that which a-factor adopts in this membrane. However, studies of the intrinsic fluorescence of the Trp residues of these peptides indicate that this residue is more deeply imbedded into the bilayer in the case of the farnesylated peptide. The a-factor is more effective in raising the bilayer to the hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine than is the S-methyl-a-factor. This bilayer-stabilizing ability is also reflected in a-factor inhibiting leakage from vesicles of N-methyldioleoylphosphatidylethanolamine. Studies on a-factor analogs permit the conclusion that the bilayer-stabilizing effect of a-factor is not solely a consequence of its greater partitioning into the membrane but is also a consequence of the degree of penetration into the bilayer and the specific conformation of the peptide at the membrane interface. These results indicate that the farnesyl group alone, in the absence of cellular factors, bestows a particular physical interaction with membranes.

Structural Requirements for Activity of the Pheromones of Ustilago hordei

“…Peptides are also prone to aggregation, which can interfere with ionization and elution from HPLC columns. Difficulties with the detection and recovery of peptides similar to those described here have been noted (Caldwell et al, 1994;Koppitz, 1996).Yeast pheromones do not aggregate (Gounarides et al, 1991;Epand et al, 1993), but this was not known for U. hordei pheromones. Sandwich assays showed that the farnesylated MAT-2 9-mer peptide passed through dialysis membrane and induced conjugation tubes, but the active MAT-1 12-mer methyl ester of virtually the same mass did not.…”

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

“…In simultaneously performed bioassays, wild-type farnesylated a-factor exhibits endpoints of activity of 25 and 250 pg in the induction of growth arrest and mating restoration, respectively. Although such differences in pheromone bioactivity may be attributable to differences in the concentration of monomeric mating factors in the medium, this is unlikely in light of several published observations (30,40,41). Marcus et al (30) demonstrated that the biological activity ofa-factor was almost identical in three different biological assays in which endpoints of activity were determined in either solution or agar-solidified medium.…”

“…Marcus et al (30) demonstrated that the biological activity ofa-factor was almost identical in three different biological assays in which endpoints of activity were determined in either solution or agar-solidified medium. In addition, assuming the pheromone interacts with its receptor in a membranous environment, circular dichroism studies on a-factor in the presence of phospholipid vesicles have indicated that no aggregation of the pheromone occurs (40). Finally, there was no evidence of a-factor aggregation in NMR studies performed in dimethyl sulfoxide (41).…”

Synthesis of high specific activity 35S‐labelled N‐methanesulfonyl farnesylcysteine and a photoactive analog

“…[5][6][7] Prenylation is required for both normal and oncogenic biological activity of these proteins as prenylation enhances their hydrophobicity, allowing for translocation from the cytosol to membranes where further signaling events transpire. 8 Prenyltransferases catalyze the addition of an isoprenoid moiety from a prenyl diphosphate to the G-protein substrate. These enzymes have been extensively examined for their therapeutic potential and numerous inhibitors have been generated towards this end.…”

“…Several studies have suggested or confirmed the existence of isoprenoid binding sites within proteins which interact with prenylated proteins, implying a purpose for the prenyl group beyond the simple facilitation of hydrophobic interactions with membranes. 1,8,[14][15][16][17][18][19][20][21] The design of compounds which interfere with key interactions between a prenylated protein and a cognate receptor protein offers a promising, and potentially more specific, alternative to the current approaches that target inhibition of the prenylation step.…”