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

Katrine Rude Laub,Sara Abad Herrera,Gunnar Dittmar,Magdalena Marek,Thomas Günther Pomorski,Tamara Kanashova,Adèle Bourmaud,Lyubomir Dimitrov Stanchev

doi:10.1371/journal.pone.0184236

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.

Highlights

ATP binding cassette (ABC) transporters are members of a superfamily of proteins that mediate ATP-driven unidirectional transport of a variety of molecules across biological membranes
For expression analysis and purification of Pdr11p, three different 2-micron—based multicopy plasmids were constructed to allow for galactose-inducible expression: a N-terminal FLAG-tagged Pdr11p (Pdr11p), a FLAG-tagged Pdr11p fused at the C terminus with green fluorescent protein (Pdr11p-GFP), and a catalytically inactive mutant of FLAG-tagged Pdr11p (Pdr11pK788M) carrying a lysine to methionine substitution in the Walker A region of NBD2 (Table 1) known to block the ATPase activity of ABC proteins [13, 20, 21]
We describe a successful purification protocol and the biochemical characterisation of the yeast ABC transporter Pdr11p

Summary

Introduction

ATP binding cassette (ABC) transporters are members of a superfamily of proteins that mediate ATP-driven unidirectional transport of a variety of molecules across biological membranes. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Methods

Results

Conclusion