serval:BIB_A3156323E8DF
Purification and characterisation of the yeast plasma membrane ATP binding cassette transporter Pdr11p.
10.1371/journal.pone.0184236
000411059300013
28922409
Laub
K.R.
author
Marek
M.
author
Stanchev
L.D.
author
Herrera
S.A.
author
Kanashova
T.
author
Bourmaud
A.
author
Dittmar
G.
author
Günther Pomorski
T.
author
article
2017
PLoS One
1932-6203
1932-6203
journal
12
9
e0184236
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.
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
eng
60_published
true
peer-reviewed
University of Lausanne
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