The pathogen-related yeast protein Pry1, a member of the CAP protein superfamily, is a fatty acid-binding protein

Rabih Darwiche,Laurent Mène-Saffrané,David Gfeller,Oluwatoyin A Asojo,Roger Schneiter

doi:10.1074/jbc.m117.781880

Rabih Darwiche, Laurent Mène-Saffrané + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m117.781880

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Abstract

Members of the CAP superfamily (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins), also known as SCP superfamily (sperm-coating proteins), have been implicated in many physiological processes, including immune defenses, venom toxicity, and sperm maturation. Their mode of action, however, remains poorly understood. Three proteins of the CAP superfamily, Pry1, -2, and -3 (pathogen related in yeast), are encoded in the Saccharomyces cerevisiae genome. We have shown previously that Pry1 binds cholesterol in vitro and that Pry function is required for sterol secretion in yeast cells, indicating that members of this superfamily may generally bind sterols or related small hydrophobic compounds. On the other hand, tablysin-15, a CAP protein from the horsefly Tabanus yao, has been shown to bind leukotrienes and free fatty acids in vitro Therefore, here we assessed whether the yeast Pry1 protein binds fatty acids. Computational modeling and site-directed mutagenesis indicated that the mode of fatty acid binding is conserved between tablysin-15 and Pry1. Pry1 bound fatty acids with micromolar affinity in vitro, and its function was essential for fatty acid export in cells lacking the acyl-CoA synthetases Faa1 and Faa4. Fatty acid binding of Pry1 is independent of its capacity to bind sterols, and the two sterol- and fatty acid-binding sites are nonoverlapping. These results indicate that some CAP family members, such as Pry1, can bind different lipids, particularly sterols and fatty acids, at distinct binding sites, suggesting that the CAP domain may serve as a stable, secreted protein domain that can accommodate multiple ligand-binding sites.

Highlights

Members of the CAP superfamily, known as SCP superfamily, have been implicated in many physiological processes, including immune defenses, venom toxicity, and sperm maturation
Fatty acid binding of Pry1 is independent of its capacity to bind sterols, and the two sterol- and fatty acid-binding sites are nonoverlapping. These results indicate that some CAP family members, such as Pry1, can bind different lipids, sterols and fatty acids, at distinct binding sites, suggesting that the CAP domain may serve as a stable, secreted protein domain that can accommodate multiple ligand-binding sites
Tablysin-15 binds leukotrienes as well as free palmitic acid by a lipid-binding channel formed between the two parallel running helices, ␣1 and ␣3, and closed at the bottom by a much shorter helix ␣4, which runs perpendicular to helices ␣1 and ␣3 [12]

Summary

Results and discussion

Tablysin-15 binds leukotrienes as well as free palmitic acid by a lipid-binding channel formed between the two parallel running helices, ␣1 and ␣3, and closed at the bottom by a much shorter helix ␣4, which runs perpendicular to helices ␣1 and ␣3 [12]. Expression of tablysin-15 in cells lacking Pry function failed to complement the block in sterol export, confirming the results of the in vitro binding assay (Fig. 3, B and C) Taken together, these results indicate that Pry has at least two independent lipid-binding sites, the caveolin-binding motif, which is required for binding free sterols, and the hydrophobic binding channel between helices ␣1 and ␣3, which constitutes the binding site for free fatty acids and leukotrienes. We generated a strain in which the transcription of PRY1 could be shut off through a switch in carbon sources Cultivation of this strain, bearing a plasmid-borne copy of PRY1 under control of a galactose-inducible and glucose-repressible promoter, in a background lacking genomic Pry function as well as the two acyl-CoA synthetases (pGALPRY1 pry1⌬ pry2⌬ pry3⌬ faa1⌬ faa4⌬), in glucose media resulted in a time-dependent increase of intracellular free fatty acids (Fig. 8B). This ligand-binding CAP domain could be further functionalized by the addition of auxiliary modules, such as the arginine-glycine-aspartic acid (RGD) motif, which is present in tablysin-15 and binds integrins [11], or the potassium ion channel inhibitor-like fold, which is present in the cysteine-rich domain of stecrisp and the mouse testis specific protein 1 (Tpx-1) [28, 29]

Experimental procedures

Lipid labeling and analysis

Fatty acid quantification

In vitro lipid binding assay