Phosphatidylinositol binding of Saccharomyces cerevisiae Pdr16p represents an essential feature of this lipid transfer protein to provide protection against azole antifungals - PubMed (original) (raw)

Fig. 3

Comparison of Pdr16p and its mutant defective in PI binding, Pdr16p E235A, K267A. A: Pdr16p and Pdr16p E235A, K267A can be stably expressed in E. coli and purified using Ni-NTA beads. SDS-PAGE gel of indicated amounts of purified recombinant Pdr16 and Pdr16 E235A, K267A proteins was stained with Coomassie blue. B: Both, Pdr16p and Pdr16p E235A, K267A are present as homodimers in E. coli extracts. The purified recombinant Pdr16 proteins were subjected to size exclusion chromatography on a Superose™ 12 10/300 GL column, using bovine serum albumin (BSA) and ovalbumin as protein molecular weight standards. Both Pdr16 proteins elute with an apparent molecular mass of approximately 90 kDa. Predicted molecular mass of Pdr16p monomer is 40.7 kDa. C: Lipid–protein overlays using wild-type Pdr16p and Pdr16p E235A, K267A, respectively, showed the preferred affinity of both proteins to PA and PI4P immobilized on the nitrocellulose membranes. Abbreviations: LPA, Lysophosphatidic acid; LPC, Lysophosphocholine; PI, Phosphatidylinositol; PI4P, Phosphatidylinositol (4) phosphate; PI3P, Phosphatidylinositol (3) phosphate; PI5P, Phosphatidylinositol (5) phosphate; PE, Phosphatidylethanolamine; PC, Phosphatidylcholine; S1P, Sphingosine 1-Phosphate; PI3,4P2, Phosphatidylinositol (3,4) bisphosphate; PI3,5P2, Phosphatidylinositol (3,5) bisphosphate; PI4,5P2, Phosphatidylinositol (4,5) bisphosphate; PI3,4,5P3, Phosphatidylinositol (3,4,5) trisphosphate; PA, Phosphatidic acid; PS, Phosphatidylserine; Blank, no lipid spotted.