Purification and characterisation of the yeast plasma membrane ATP binding cassette transporter Pdr11p
| Autor: | Adèle Bourmaud, Magdalena Marek, Gunnar Dittmar, Katrine Rude Laub, Sara Abad Herrera, Thomas Günther Pomorski, Tamara Kanashova, Lyubomir Dimitrov Stanchev |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
Adenosine Triphosphatase
0301 basic medicine 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 Cell Membranes lcsh:Medicine Yeast and Fungal Models ATP-binding cassette transporter Biochemistry ATP hydrolysis lcsh:Science Multidisciplinary biology Chemistry Walker motifs Lipids Recombinant Proteins Enzymes Sterols Experimental Organism Systems Cellular Structures and Organelles Technology Platforms Research Article Saccharomyces cerevisiae Proteins Protein domain Saccharomyces cerevisiae Research and Analysis Methods Saccharomyces 03 medical and health sciences Model Organisms FLAG-tag Vesicles Cell Membrane lcsh:R Phosphatases Organisms Fungi Biology and Life Sciences Proteins Membrane Proteins Transporter Cell Biology biology.organism_classification Yeast 030104 developmental biology Membrane protein Liposomes Enzymology ATP-Binding Cassette Transporters lcsh:Q Vanadates |
| Zdroj: | PLoS One, vol. 12, no. 9, pp. e0184236 Laub, K R, Marek, M, Stanchev, L D, Herrera, S A, Kanashova, T, Bourmaud, A, Dittmar, G & Pomorski, T G 2017, ' Purification and characterisation of the yeast plasma membrane ATP binding cassette transporter Pdr11p ', PLOS ONE, vol. 12, no. 9, e0184236 . https://doi.org/10.1371/journal.pone.0184236 PLoS ONE, Vol 12, Iss 9, p e0184236 (2017) PLoS ONE |
| DOI: | 10.1371/journal.pone.0184236 |
| Popis: | The ATP binding cassette (ABC) transporters Pdr11p and its paralog Aus1p are expressed under anaerobic growth conditions at the plasma membrane of the yeast \(\textit {Saccharomyces cerevisiae}\) 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. |
| Databáze: | OpenAIRE |
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