Article: article from journal or magazin.
Purification and characterisation of the yeast plasma membrane ATP binding cassette transporter Pdr11p.
The ATP binding cassette (ABC) transporters Pdr11p and its paralog Aus1p are expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and are required for sterol uptake. However, the precise mechanism by which these ABC transporters facilitate sterol movement is unknown. In this study, an overexpression and purification procedure was developed with the aim to characterise the Pdr11p transporter. Engineering of Pdr11p variants fused at the C terminus with green fluorescent protein (Pdr11p-GFP) and containing a FLAG tag at the N terminus facilitated expression analysis and one-step purification, respectively. The detergent-solubilised and purified protein displayed a stable ATPase activity with a broad pH optimum near 7.4. Mutagenesis of the conserved lysine to methionine (K788M) in the Walker A motif abolished ATP hydrolysis. Remarkably, and in contrast to Aus1p, ATPase activity of Pdr11p was insensitive to orthovanadate and not specifically stimulated by phosphatidylserine upon reconstitution into liposomes. Our results highlight distinct differences between Pdr11p and Aus1p and create an experimental basis for further biochemical studies of both ABC transporters to elucidate their function.
ATP-Binding Cassette Transporters/biosynthesis, ATP-Binding Cassette Transporters/chemistry, ATP-Binding Cassette Transporters/genetics, ATP-Binding Cassette Transporters/isolation & purification, Cell Membrane/chemistry, Cell Membrane/genetics, Cell Membrane/metabolism, Gene Expression, Liposomes/chemistry, Protein Domains, Recombinant Proteins/biosynthesis, Recombinant Proteins/chemistry, Recombinant Proteins/genetics, Recombinant Proteins/isolation & purification, Saccharomyces cerevisiae/chemistry, Saccharomyces cerevisiae/genetics, Saccharomyces cerevisiae/metabolism, Saccharomyces cerevisiae Proteins/biosynthesis, Saccharomyces cerevisiae Proteins/chemistry, Saccharomyces cerevisiae Proteins/genetics, Saccharomyces cerevisiae Proteins/isolation & purification, Vanadates/chemistry
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