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Purification and characterisation of the yeast plasma membrane ATP binding cassette transporter Pdr11p

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Item Type:Article
Title:Purification and characterisation of the yeast plasma membrane ATP binding cassette transporter Pdr11p
Creators Name:Laub, K.R. and Marek, M. and Stanchev, L.D. and Herrera, S.A. and Kanashova, T. and Bourmaud, A. and Dittmar, G. and Guenther Pomorski, T.
Abstract: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.
Keywords:ATP-Binding Cassette Transporters, Cell Membrane, Gene Expression, Liposomes, Protein Domains, Recombinant Proteins, Saccharomyces Cerevisiae, Saccharomyces Cerevisiae Proteins, Vanadates
Source:PLoS ONE
Publisher:Public Library of Science
Page Range:e0184236
Date:18 September 2017
Official Publication:https://doi.org/10.1371/journal.pone.0184236
PubMed:View item in PubMed

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