Solubilisation and purification of Pdr11p from <i>hem1</i>Δ<i>aus1</i>Δ<i>pdr11</i>Δ cells.

<p>A: Flowchart of the procedure. Supernatant S1 was centrifuged at 10,000 or 60,000 g (45 min) for collection of plasma membrane enriched fractions or total yeast membranes, respectively. B: Solubilisation of Pdr11p from total yeast membranes. Equal amounts of membranes (P2) were solubilised at the given DDM concentrations. After ultracentrifugation, the amounts of solubilised and non-solubilised Pdr11p were estimated in aliquots of supernatants (S3) and pellets (P3), respectively, via immunoblotting with anti-FLAG antibodies. Pdr11p, filled arrowheads; breakdown product, open arrowhead. C, original membrane fraction not been subjected to solubilisation (positive control). C: Purification of Pdr11p from plasma membrane enriched fractions. Representative Western blot analysis with anti-FLAG antibodies and Coomassie stained SDS-PAGE (SDS) of selected purification fractions. The loaded purification fractions are normalised with respect to volume. Sizes of the molecular mass standards are indicated on the sides.</p

Expression analysis utilising GFP-tagged Pdr11p.

<p>A: Effect of induction time on Pdr11p-GFP expression in <i>hem1</i>Δ<i>aus1</i>Δ<i>pdr11</i>Δ. Panels I-IV, Representative flow cytometry based histograms of cells transformed with plasmids carrying <i>PDR11-GFP</i> (green) or empty vector (black) after 6 and 17 h in SD medium (non-induced) or SG medium (induced). Each data set consists of minimum 20,000 cells. Panels V-VIII, Representative fluorescence microscopy images of cells expressing Pdr11p-GFP after 6 and 17 h induction. The intensities of the fluorescent signals cannot be compared between the images. Bars equal 10 <i>μ</i>m. B: Percentage of cells expressing Pdr11p-GFP after galactose induction as a function of optical density at 600 nm (OD₆₀₀) and cell concentration of the glucose pre-culture just prior to media change. All cultures in each repetition grew from a single colony. Lines are included solely to guide the eye. <i>hem1</i>Δ<i>aus1</i>Δ<i>pdr11</i>Δ strain, expression after 6 h induction in 3 cultures from the same selected high-expressing colony. BJ1991 strain, expression after 8 (open triangles) and 10 h (open diamonds) from the same selected colony.</p

ATPase activity of solubilised Pdr11p.

<p>ATPase activity of the purified detergent-solubilised transporter was assayed as described under “Materials and Methods” using [<i>γ</i>-³²P] ATP. A: ATPase activity as a function of pH. Open and filled circles are data from two independent experiments. Values are normalised with respect to the values at pH 7.2 (open circles) or pH 7.4 (closed circles). The dashed line is included to guide the eye. B: Effect of various inhibitors: NaN₃, 5 mM; ouabain, 5 mM; BeSO₄, 1 mM; NaF, 5 mM; AlF₃, 1 mM; orthovanadate, 1 mM; EDTA, 1 mM. C: ATPase activity as a function of orthovanadate concentration. Fitting of data to a dose-response/activity curve (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184236#sec002" target="_blank">Material and methods</a>) gives <i>IC</i>₅₀ = 4 ± 2 mM, and a Hill coefficient = 0.8 ± 0.2. Results in B and C are the mean ± S.D. from at least two independent experiments relative to the value obtained for the purified detergent-solubilised protein in the absence of inhibitors (control).</p

ATPase activity of liposome-reconstituted Pdr11p and Aus1p.

<p>Purified Pdr11p and Aus1p were reconstituted into different liposomes (containing Rho-PE as fluorescent lipid marker) and assayed for ATPase activity using [<i>γ</i>-³²P] ATP. A: SDS PAGE analysis of a flotation assay of Pdr11p proteoliposomes in a sucrose gradient. Detection of lipids and protein in the same low density fraction validated successful reconstitution. Proteins are visualised by silver staining and lipids by fluorescence from Rho-PE. B: Relative ATPase activity of Pdr11p reconstituted in PS liposomes in presence of the indicated inhibitors: orthovanadate, 1 mM; BeSO₄, 1 mM; NaF, 5 mM. Data is based on at least two reconstitutions from one purification batch. C: Lipid effect on ATPase activity of reconstituted Pdr11p and Aus1p. All activities are corrected for protein amount in the proteoliposomes. Data is based on two reconstitutions from one purification batch of each protein. PC, PC only; PS, PC/PS (1:1); PG, PC/PG (7:3).</p

Effect of various compounds on ATPase activity of purified Pdr11p^a.

<p>Effect of various compounds on ATPase activity of purified Pdr11p<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184236#t002fn001" target="_blank">^a</a>.</p

Overexpression and functionality of tagged Pdr11p variants.

<p><i>S. cerevisiae</i> cells were transformed with empty vector (e.v.) or pESC-URA carrying <i>PDR11</i>, <i>PDR11-GFP</i>, or <i>PDR11^K788M</i>. Sizes of the molecular mass standards are given to the left when relevant. A: Western blot illustrating the induced expression of FLAG-tagged variants of Pdr11p (filled arrowheads) and breakdown product (open arrowhead). Cells carrying the empty vector served as control. The blot was stained with anti-FLAG antibody. B: In-gel fluorescence detection using Criterion TGX stain-free gel. C: Serial dilutions of transformed heme-deficient <i>hem1</i>Δ and sterol uptake-deficient triple mutant <i>hem1</i>Δ<i>aus1</i>Δ<i>pdr11</i>Δ strains spotted onto standard synthetic galactose plates supplemented with <i>δ</i>-aminolevulinic acid (ALA) or cholesterol.</p

Conserved ABC motifs of Aus1p and Pdr11p.

<p>Conserved ABC motifs of Aus1p and Pdr11p.</p

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 $\textit {Saccharomyces cerevisiae}$ 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