Cholesterol-Binding by the Yeast CAP Family Member Pry1 Requires the Presence of an Aliphatic Side Chain on Cholesterol

Abstract

Pathogen-related yeast protein 1 (Pry1) is a Saccharomyces cerevisiae member of the CAP/SCP/TAPS superfamily. Although, CAP proteins have been proposed to be implicated in a number of physiological processes, such as pathogen virulence, sperm maturation and fertilization, host-pathogen interactions and defense mechanisms, the molecular mode of action of these proteins is poorly understood. CAP proteins are mostly secreted and they are stable in the extracellular space over a wide a range of conditions. All members of this superfamily contain a common CAP domain of approximately 150 amino acids, which adopts a unique α-β-α sandwich fold. We have previously shown that the yeast CAP family members act as sterol-binding and -export proteins in vivo and that the Pry proteins bind cholesterol and cholesteryl acetate in vitro. The conserved CAP domain of Pry1 is necessary and sufficient for sterol binding. Based on these observations, it is conceivable that CAP proteins exert their biological function through a common mechanism, such as binding and sequestration of sterols or related small hydrophobic compounds. Here we analyze the ligand specificity of Pry1 in more detail and show that the presence of the aliphatic isooctane side chain of the sterol but not the 3-hydroxyl group is important for binding to Pry1.

The function of yeast CAP family proteins in lipid export, mating, and pathogen defense

“…Eugenol competes with cholesterol for binding to Pry1 and Pry2, indicating that the binding sites for the two lipids are overlapping, which is consistent with molecular modeling studies (Fig. C,D) . These data thus indicate that CAP proteins might not only promote the secretion of potentially harmful hydrophobic compounds, such as, for example, intermediates in sterol biosynthesis, but they might also sequester such compounds when present in the extracellular space .…”

Plant pathogenesis–related proteins of the cacao fungal pathogen Moniliophthora perniciosa differ in their lipid-binding specificities

“…Such a possible mode of action of CAP proteins is supported by the observation that yeast mutants lacking Pry function are hypersensitive to eugenol, a member of the alkylbenzene class of compounds present in clove oil, nutmeg, cinnamon, and bay leaf, used as local antiseptic and anesthetic. Importantly, this eugenol sensitivity is proportional to expression levels of Pry proteins and eugenol competes with cholesterol for binding to Pry in vitro, indicating that the flexible loop harboring the CBM can not only bind sterols but also other small hydrophobic compounds such as for example eugenol, thereby protecting cells from its toxicity (18,23). A possible sequestrationbased mode of action of CAP family members in plant innate immunity is supported by the fact that plant PR-1 binds sterols in vitro and addition of the purified PR-1 protein inhibits growth of the sterol-auxotrophic plant pathogenic oomycete Phytophthora (21).…”

“…Expression of tablysin-15, however, does not rescue the sterol export phenotype but rescues export of fatty acids (19). The sterol and fatty acid binding and export function of the yeast CAP proteins is confined to the CAP domain, because expression of the CAP domain alone is sufficient to rescue the sterol and fatty acid export phenotype of a pry1 pry2 double mutant, and the CAP domain of Pry1 alone binds sterols and fatty acids at distinct sites in vitro (19,22,23). Sterol binding by Pry proteins requires the displacement of a flexible loop containing aromatic amino acids, termed the caveolin-binding motif (CBM) within the CAP domain, since point mutations within this motif abolish sterol export and binding, whereas fatty acid binding takes place in the groove between two parallel running helices, 1 and 3 (19,24).…”

“…Sterol binding by Pry proteins requires the displacement of a flexible loop containing aromatic amino acids, termed the caveolin-binding motif (CBM) within the CAP domain, since point mutations within this motif abolish sterol export and binding, whereas fatty acid binding takes place in the groove between two parallel running helices, 1 and 3 (19,24). Taken together, these data indicate that CAP proteins may exert an immunomodulatory function through binding hydrophobic ligands, such as sterols or related small hydrophobic compounds and fatty acids and their derivatives such as leukotrienes (19,23,25).…”

Protein‐facilitated transport of hydrophobic molecules across the yeast plasma membrane

“…This putative mode of sterol binding diverges from what is seen in cytosolic sterol‐binding proteins, such as Osh4, StAR/STARD1, or NPC2, where substrate‐binding site is buried in a hydrophobic tunnel inside the protein. A variety of sterols, steroids and sterol precursors were shown to bind Pry1 with competitive efficiency . For a complete block in cholesterol acetate secretion, a double knockout of Pry1 and Pry2 was necessary, which indicates their redundant activity.…”