TY - JOUR
T1 - Purification and characterisation of the yeast plasma membrane ATP binding cassette transporter Pdr11p
AU - Laub, Katrine Rude
AU - Marek, Magdalena
AU - Stanchev, Lyubomir Dimitrov
AU - Herrera, Sara Abad
AU - Kanashova, Tamara
AU - Bourmaud, Adèle
AU - Dittmar, Gunnar
AU - Pomorski, Thomas Günther
N1 - Publisher Copyright:
© 2017 Laub et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2017/9
Y1 - 2017/9
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85029580865&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0184236
DO - 10.1371/journal.pone.0184236
M3 - Article
C2 - 28922409
AN - SCOPUS:85029580865
SN - 1932-6203
VL - 12
JO - PLoS ONE
JF - PLoS ONE
IS - 9
M1 - e0184236
ER -