| Autor: |
Laub KR; Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg C, Denmark., Marek M; Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg C, Denmark., Stanchev LD; Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg C, Denmark.; Department of Molecular Biochemistry, Ruhr University Bochum, Bochum, Germany., Herrera SA; Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg C, Denmark.; Department of Molecular Biochemistry, Ruhr University Bochum, Bochum, Germany., Kanashova T; Mass spectrometry core unit, Max Delbrück Center for Molecular Medicine, Berlin, Germany., Bourmaud A; Proteome and Genome research laboratory, Luxembourg Institute of Health, Strassen, Luxembourg., Dittmar G; Mass spectrometry core unit, Max Delbrück Center for Molecular Medicine, Berlin, Germany.; Proteome and Genome research laboratory, Luxembourg Institute of Health, Strassen, Luxembourg., Günther Pomorski T; Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg C, Denmark.; Department of Molecular Biochemistry, Ruhr University Bochum, Bochum, Germany. |
| Abstrakt: |
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. |
