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 m...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2017-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC5602531?pdf=render |
| _version_ | 1818000481916551168 |
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| author | Katrine Rude Laub Magdalena Marek Lyubomir Dimitrov Stanchev Sara Abad Herrera Tamara Kanashova Adèle Bourmaud Gunnar Dittmar Thomas Günther Pomorski |
| author_facet | Katrine Rude Laub Magdalena Marek Lyubomir Dimitrov Stanchev Sara Abad Herrera Tamara Kanashova Adèle Bourmaud Gunnar Dittmar Thomas Günther Pomorski |
| author_sort | Katrine Rude Laub |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-14T03:22:11Z |
| format | Article |
| id | doaj.art-e5215dc6bf7040e284cf948303dcf867 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-14T03:22:11Z |
| publishDate | 2017-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-e5215dc6bf7040e284cf948303dcf8672022-12-22T02:15:17ZengPublic Library of Science (PLoS)PLoS ONE1932-62032017-01-01129e018423610.1371/journal.pone.0184236Purification and characterisation of the yeast plasma membrane ATP binding cassette transporter Pdr11p.Katrine Rude LaubMagdalena MarekLyubomir Dimitrov StanchevSara Abad HerreraTamara KanashovaAdèle BourmaudGunnar DittmarThomas Günther PomorskiThe 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.http://europepmc.org/articles/PMC5602531?pdf=render |
| spellingShingle | Katrine Rude Laub Magdalena Marek Lyubomir Dimitrov Stanchev Sara Abad Herrera Tamara Kanashova Adèle Bourmaud Gunnar Dittmar Thomas Günther Pomorski Purification and characterisation of the yeast plasma membrane ATP binding cassette transporter Pdr11p. PLoS ONE |
| title | Purification and characterisation of the yeast plasma membrane ATP binding cassette transporter Pdr11p. |
| title_full | Purification and characterisation of the yeast plasma membrane ATP binding cassette transporter Pdr11p. |
| title_fullStr | Purification and characterisation of the yeast plasma membrane ATP binding cassette transporter Pdr11p. |
| title_full_unstemmed | Purification and characterisation of the yeast plasma membrane ATP binding cassette transporter Pdr11p. |
| title_short | Purification and characterisation of the yeast plasma membrane ATP binding cassette transporter Pdr11p. |
| title_sort | purification and characterisation of the yeast plasma membrane atp binding cassette transporter pdr11p |
| url | http://europepmc.org/articles/PMC5602531?pdf=render |
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