
==== Front
Protein Sci
Protein Sci
10.1002/(ISSN)1469-896X
PRO
Protein Science : A Publication of the Protein Society
0961-8368
1469-896X
John Wiley & Sons, Inc. Hoboken, USA

39312388
10.1002/pro.5181
PRO5181
Research Article
Research Article
An in vitro set‐up to study Pdr5‐mediated substrate translocation
Gala Marti et al.
Gala Marti Stefanie L. 1
Wagner Manuel 1 2
Nentwig Lea‐Marie 1
Smits Sander H. J. 1 3
Schmitt Lutz https://orcid.org/0000-0002-1167-9819
1 lutz.schmitt@hhu.de

1 Institute of Biochemistry Heinrich Heine University Düsseldorf Düsseldorf Germany
2 OQEMA GmbH Mönchengladbach Germany
3 Center for Structural Studies Heinrich Heine University Düsseldorf Düsseldorf Germany
* Correspondence
Lutz Schmitt, Institute of Biochemistry, Heinrich Heine University Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany.
Email: lutz.schmitt@hhu.de

23 9 2024
10 2024
23 9 2024
33 10 10.1002/pro.v33.10 e518123 7 2024
14 5 2024
06 9 2024
© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

Abstract

Pdr5 is the most abundant ABC transporter in Saccharomyces cerevisiae and plays a major role in the pleiotropic drug resistance (PDR) network, which actively prevents cell entry of a large number of structurally unrelated compounds. Due to a high level of asymmetry in one of its nucleotide binding sites (NBS), Pdr5 serves as a perfect model system for asymmetric ABC transporter such as its medical relevant homologue Cdr1 from Candida albicans. In the past 30 years, this ABC transporter was intensively studied in vivo and in plasma membrane vesicles. Nevertheless, these studies were limited since it was not possible to isolate and reconstitute Pdr5 in a synthetic membrane system while maintaining its activity. Here, the functional reconstitution of Pdr5 in a native‐like environment in an almost unidirectional inside‐out orientation is described. We demonstrate that reconstituted Pdr5 is capable of translocating short‐chain fluorescent NBD lipids from the outer to the inner leaflet of the proteoliposomes. Moreover, this transporter revealed its ability to utilize other nucleotides to accomplish transport of substrates in a reconstituted system. Besides, we were also able to estimate the NTPase activity of reconstituted Pdr5 and determine the kinetic parameters for ATP, GTP, CTP, and UTP.

NBD lipids
NTPase activity
Pdr5
protein reconstitution
transport activity
Deutsche Forschungsgemeinschaft 10.13039/501100001659 Schm1279/17‐1 417919780 source-schema-version-number2.0
cover-dateOctober 2024
details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_JATSPMC version:6.4.8 mode:remove_FC converted:23.09.2024
Gala Marti SL , Wagner M , Nentwig L‐M , Smits SHJ , Schmitt L . An in vitro set‐up to study Pdr5‐mediated substrate translocation. Protein Science. 2024;33 (10 ):e5181. 10.1002/pro.5181 39312388

Review Editor: Aitziber L. Cortajarena.
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pmc1 INTRODUCTION

The ATP‐binding cassette (ABC) transporter family comprise one of the major superfamilies of membrane transport proteins and can be found in all domains of life (Holland et al., 2003). While exporter were found in all organisms, importer were only found in prokaryotes with only a handful of exceptions in eukaryotes (Choi & Ford, 2021; Oswald et al., 2006). Nevertheless, they all share a common architecture of two transmembrane domains (TMDs) and two nucleotide‐binding domains (NBDs). The latter bind and hydrolyze ATP, providing energy for the active transport of substrates across the membrane (Schmitt & Tampe, 2002). Notably, overexpression of these proteins often leads to a phenomenon referred to as multi‐drug resistance (MDR). This phenomenon describes the ability of organisms to develop resistance against a large number of structurally and functionally unrelated compounds. As a consequence, it is one of the major obstacles in the treatment of fungal pathogenic infections, bacterial infections and cancer (Ernst et al., 2005; Gottesman et al., 2002; Lage, 2003).

Pdr5 from Saccharomyces cerevisiae is one of the most abundant and well‐studied pleiotropic drug resistance (PDR) transporter. Playing a crucial role in the PDR network, Pdr5 confers resistance to a wide range of drugs, which are chemically different but have a similar range of molecular volume (Golin & Ambudkar, 2015).

For more than 30 years, Pdr5 has been the focus of extensive research to understand the molecular mechanisms of drug export (Golin & Ambudkar, 2015). Pdr5 is a full‐size transporter, consisting of two transmembrane domains (TMDs) and two nucleotide‐binding domains (NBDs), arranged in a reverse topology starting with the first NBD at the N‐terminus. As characteristic feature of ABC transporters from the PDR subfamily, Pdr5 has a flexible N‐terminal extension of ~130 amino acids of unknown function (Golin & Ambudkar, 2015; Lamping et al., 2010). Due to a high degree of substitutions in key residues of the distinct motifs in one nucleotide‐binding site, Pdr5 belongs to the family of asymmetric ABC transporters and serves as a model system for other asymmetric transporters (Stockner et al., 2020), for example, CFTR or ABCG5/G8. Even though the deviant nucleotide‐binding site, NBS1, can bind ATP, it is not capable of hydrolyzing this nucleotide (Ernst et al., 2008; Gupta et al., 2014; Harris et al., 2021; Wagner et al., 2019).

Recently, structures of Pdr5 were published, providing new insights into structural features and protein function (Harris et al., 2021). For example, sequence alignment of the PDR subfamily revealed a highly conserved motif ‘MQKGEIL’ within the linker domain, contacting the nucleotide bound to the deviant nucleotide‐binding site of Pdr5. A mutational study demonstrated a crucial structural role of this motif with a complex, substrate‐dependent pattern (Harris et al., 2021). Moreover, in another study, the four residues comprising the highly asymmetric efflux gate of Pdr5 were analyzed, revealing the importance of the residues Gly‐682, Phe‐683, Gly‐1371, and Met‐1373 in molecular gating, allosteric regulation, conformational switching, and protein folding (Alhumaidi et al., 2022).

Nevertheless, functional studies were so far limited to experiments in vivo or with highly enriched Pdr5 plasma membrane vesicles. Only recently, a functional purification was described (Wagner et al., 2019), but so far, it was not possible to characterize isolated and purified Pdr5 in a membrane‐like environment. Within the past years, reconstitution of proteins into liposomes has emerged as a valuable experimental approach to investigate their functional properties and gain mechanistic insights (Shen et al., 2013). The process of reconstitution involves the incorporation of purified Pdr5 into synthetic lipid vesicles that mimic the natural lipid bilayer composition. This approach offers a controlled and well‐defined system to study the ATPase and transport activity of Pdr5 in a membrane‐like environment.

By comprehensively studying Pdr5 in liposomes, we aim to gain deeper insights into the structure–function relationship and mechanistic aspects of this PDR transporter. In this study, we demonstrate for the first time the successful reconstitution of active Pdr5 into liposomes and investigate its ATPase activity and transport function.

2 RESULTS

2.1 Efficiency of Pdr5 reconstitution into liposomes

Pdr5 was overexpressed in S. cerevisiae and purified via IMAC and SEC following the previously described protocol (Wagner et al., 2019). Homogenous samples of wildtype Pdr5 (Pdr5 WT) and the Pdr5 E1036Q (Pdr5 EQ) mutant were used for reconstitution into liposomes composed of a mixture of POPC, POPE, POPS, and ergosterol (van't Klooster et al., 2020). The liposomes were destabilized stepwise with Triton X‐100 until the absorption at 540 nm decreased to 60%. Under these conditions, it was anticipated that Pdr5 would conceivably be incorporated into the liposomes in a preferred inside‐out orientation. For monitoring the total amount of incorporated protein, a quantitative SDS‐PAGE analysis was conducted (Figure 1a).

FIGURE 1 Reconstitution efficiency and orientation of Pdr5 in liposomes. (a) Quick Coomassie stained quantitative SDS‐PAGE of purified Pdr5 samples with known concentrations and 5‐fold concentrated proteoliposomes sample of unknown concentration. Molecular weight of marker protein is given in kDa to the left. (b) Quantitative analysis of the total protein incorporation into liposomes and (c) the amount of Pdr5 in an inside‐out orientation in percentage. The error bars represent the mean ± SE of three independent measurements.

The intensity of a sample containing proteoliposomes was compared to the intensity of samples containing purified protein with known concentrations. The described reconstitution protocol yielded a total protein concentration of 0.023 ± 0.001 mg/ml, corresponding to an incorporation efficiency of 58 ± 1.5%. The orientation of Pdr5 in liposomes was determined with a digestion experiment and analyzed via a quantitative SDS‐PAGE (Figure 1a). Generally, there are two possible orientations for Pdr5 incorporation into the membrane: an inside‐out orientation, in which the NBDs are accessible from the external environment, and an inside‐in orientation, in which the NBDs are protected inside the liposomes. Addition of trypsin facilitates the digestion of the domains that are not shielded by the protecting lipid bilayer.

Considering the two nucleotide‐binding domains of Pdr5 constitute half of the proteins size, an inside‐out orientation of the protein would result in a decrease of the signal intensity at 170 kDa on SDS‐PAGE gel. The signal intensity at 170 kDa of the two samples, proteoliposomes with and without addition of trypsin, was compared to calculate the amount of Pdr5 in an inside‐out orientation. The incorporation into liposomes was 79 ± 13.8% in an inside‐out orientation (Figure 1b).

2.2 NTPase activity of proteoliposomes

Liposomes containing reconstituted Pdr5 WT were used to analyze the NTPase activity via an enzyme‐coupled assay. The hydrolysis of NTPs is stoichiometrically coupled to the oxidation of NADH and was monitored at 340 nm. Here, Pdr5 specific NTPase activity was calculated in consideration of the reconstitution efficiency and inside‐out orientation of the prepared proteoliposomes. For Pdr5 WT, a maximum ATPase activity of 748.9 ± 41.1 nmol/(min*mg) with a K m of 0.34 ± 0.06 mM was measured. Previous studies already demonstrated a significantly high basal activity and the capability of Pdr5 in plasma membrane vesicles and in detergent micelles to hydrolyze other nucleotides than ATP (Decottignies et al., 1994; Ernst et al., 2008; Golin et al., 2007; Wagner et al., 2019).

In accordance with expections, Pdr5 WT reconstituted into liposomes was able to hydrolyze also CTP, GTP, and UTP besides ATP as energy source (Figure 2 and Table 1). Although the K m for ATP and CTP were similar, the maximum activity using the latter is surprisingly higher. CTP showed the highest v max of 1202.2 ± 59.1 nmol/(min*mg) as well as the highest efficiency (v max/K m of 4.6*10−3 L/min/mg) followed by ATP (v max/K m of 2.2*10−3 L/min/mg), UTP (v max/K m of 0.8*10−3 L/min/mg) and the lowest efficiency was observed for GTP (v max/K m of 0.5*10−3 L/min/mg). UTP and GTP showed the lowest activity with 466.1 ± 43.0 nmol/(min*mg) and 262.9 ± 13.2 nmol/(min*mg), respectively.

FIGURE 2 NTPase activity of Pdr5 WT reconstituted into liposomes. (a) ATPase activity of Pdr5 proteoliposomes. (b) GTPase activity of Pdr5 proteoliposomes. (c) CTPase activity of Pdr5 proteoliposomes. (d) UTPase activity of Pdr5 proteoliposomes. The NTPase activity was measured via an enzyme‐coupled assay where the NTP hydrolysis is stoichiometrically coupled to the oxidation of NADH. The concentration of NTPs ranged from 0 to 2 mM and the NADH absorption was monitored for 40 min at 340 nm. The error bars represent the mean ± SE of three independent measurements.

TABLE 1 Kinetic parameters of Pdr5 WT NTPase activity in proteoliposomes.

NTP	v max (nmol/[min*mg])	K m (mM)	v max/K m (10−3 L/[min*mg])	k cat/K m (1/[s*mM])	
ATP	748.9 ± 41.1	0.34 ± 0.06	2.2	2.13	
GTP	262.9 ± 13.2	0.56 ± 0.08	0.5	0.75	
CTP	1202.2 ± 59.1	0.26 ± 0.04	4.6	3.42	
UTP	466.1 ± 43.0	0.57 ± 0.13	0.8	1.33	

2.3 Pdr5 mediated translocation of fluorescently labeled phospholipids

For the ABC transporters P‐gp and Cdr1, it was already demonstrated that these proteins can utilize the energy of ATP hydrolysis to flip various NBD‐labeled lipids from one to the other leaflet of liposomes (Romsicki & Sharom, 2001; Shukla et al., 2007).

Therefore, the translocation of 06:0–06:0 NBD‐labeled lipids by proteoliposomes containing Pdr5 is tested as follows. To analyze the amount of transported lipids, a fluorescence quenching assay was used (Figure 3). The prepared samples are added to a cuvette containing 50 mM KPi buffer while monitoring the fluorescence. Due to the incorporation of NBD‐labeled lipids into the proteoliposomes, the addition of the samples results in a large increase in the fluorescence signal. Addition of sodium dithionite causes a quenching effect of the unprotected NBD‐labeled lipids in the outer leaflet of the liposome. Finally, the proteoliposomes are solubilized by adding Triton X‐100 to the sample causing a quenching effect of the former protected NBD‐labeled lipids in the inner leaflet of the liposome. Therefore, the fluorescence signal drops to background levels.

FIGURE 3 Schematic overview of the fluorescence quenching assay of 06:0–06:0 NBD‐labeled lipids in proteoliposomes. Addition of dithionite leads to a quenching effect of the unprotected NBD‐labeled lipids resulting in a decrease of the fluorescence. By adding Triton X‐100 to the sample, the proteliposomes are solubilized and the former protected lipids are quenched.

To investigate a potential flippase activity of reconstituted Pdr5, liposomes containing Pdr5 were incubated with 06:0–06:0 NBD‐PC for 20 min at 25°C to ensure incorporation into the membrane. By addition of MgATP, the active transport of NBD‐PC across the membrane was initiated and measured in a time‐dependent manner for up to 100 min (Figure 4). The quantity of NBD‐PC located in the inner leaflet increased linearly over time and reached a plateau at around 60%. In contrast, liposomes containing the ATPase deficient Pdr5 mutant E1036Q (Ernst et al., 2008; Wagner et al., 2019) exhibited no significant transport of 06:0–06:0 NBD‐PC across the membrane, confirming that the increasing amount of NBD‐PC in the inner leaflet is a result of active transport mediated by Pdr5 instead of a passive flip‐flop.

FIGURE 4 Translocation of 06:0–06:0 NBD‐PC in proteoliposomes containing either Pdr5 WT or Pdr5 E1036Q. Time course of 06:0–06:0 NBD‐PC translocation up to 100 min incubation time in presence of 4 mM ATP. The percentage of NBD‐PC in the inner leaflet was corrected by subtracting the values measured for AMP (control; see Materials and Methods). The error bars represent the mean ± SE of three independent measurements.

Since Pdr5 has the capability to hydrolyze various NTPs, we assumed that the provided energy is also suited to accomplish NBD‐PC translocation in presence of different nucleotides. For this, the translocation with ATP, GTP, CTP, UTP and AMP (control) after a reaction time of 30 min was analyzed (Figure 5a). Surprisingly, the amount of transported NBD‐PC is not directly correlated to the maximum velocity of the NTPase activity. Even though the v max of CTP is 1.5‐fold higher compared to ATP, the transport activity with CTP was reduced 2‐fold. Additionally, in the presence of ATP the highest transport activity of 20% after 30 min reaction time was measured compared to the three other nucleotides. GTP, CTP and UTP resulted consequently in a reduced transport activity of approximately 10%. In addition, the passive flip‐flop of 06:0–06:0 NBD‐PC was also analyzed by using AMP instead of the NTPs. Since AMP is not hydrolyzed by Pdr5, there will be no energy available for an active transport of NBD‐labeled lipids from one to the other leaflet of the liposome. Indeed, while using AMP, the amount of translocated NBD‐PC was only 2.5% after 30 min reaction time representing the amount of transported NBD‐PC by passive flip‐flop instead of an active transport.

FIGURE 5 Translocation of 06:0–06:0 NBD‐lipids by proteoliposomes containing Pdr5 WT using various NTPs as energy source. (a) Percentage of transported 06:0–06:0 NBD‐PC in the inner leaflet of the liposome after an incubation time of 30 min in presence of 4 mM of various NTPs or AMP (control). The error bars represent the mean ± SE of at least two independent measurements. (b) Percentage of transported 06:0–06:0 NBD‐lipids with various headgroups in the inner leaflet of the liposome after an incubation time of 30 min in presence of 4 mM ATP. The error bars represent the mean ± SE of three independent measurements.

In order to determine the specificity of the translocated lipid, four short‐chain NBD‐labeled lipids with various headgroups were also tested. For this purpose, the transport assay was conducted as described above using 4 mM ATP as energy source. Pdr5 containing liposomes were either incubated with NBD‐PC, NBD‐PA, NBD‐PE or NBD‐PG for 20 min prior to the addition of ATP. The results demonstrated roughly equal amounts of translocated NBD‐labeled lipids by Pdr5 (Figure 5b). However, NBD‐PG is translocated from the outer to the inner leaflet of the liposome in slightly higher amounts than all the other tested lipids. While only 20% of NBD‐PC is transported, approximately 27% of NBD‐PG are located in the inner leaflet of the liposome. NBD‐PA and NBD‐PE are translocated at similar levels of around 22%. Therefore, there is no evidence suggesting a preference for negatively charged or zwitterionic NBD‐labeled lipids. The lack of headgroup specificity also indicates that Pdr5 is not a lipid translocase.

2.4 Drug efflux and phospholipid translocation of proteoliposomes

The above data demonstrate the capability of Pdr5 reconstituted into liposomes to actively transport short‐chain NBD‐labeled lipids across the lipid bilayer. Next the influence of various substances on the transport activity was tested. For this purpose, the well‐known Pdr5 substrates ketoconazole and clotrimazole were tested as well as the ATPase inhibitors orthovanadate and oligomycin. Here, we analyzed whether the two tested substrates compete with NBD‐PC transport or if the substrate and lipid occupy different substrate binding sites.

For all four tested substances, typical inhibition curves were obtained showing complete inhibition in a concentration‐dependent manner of NBD‐PC transport (Figure 6). Oligomycin is a known Pdr5 inhibitor (Kolaczkowski et al., 1996). Hence, it is not surprising that oligomycin addition results in an inhibition of NBD‐PC transport across the membrane. For oligomycin and orthovanadate, K i values of 4.1 ± 0.6 μM and 42.8 ± 5.3 μM were determined, respectively. On the contrary, clotrimazole is a known transport substrate and non‐competitive inhibitor of the ATPase activity of Pdr5. This substance and it's derivates are xenobiotic compounds and are widely used as antifungal agents (Golin et al., 2007). The addition of clotrimazole led to a complete inhibition of the transport with a K i value of 8.1 ± 0.8 μM. However, already small amounts of Clotrimazole inhibited the transport activity of NBD‐PC, suggesting that this substance is transported rather than the short‐chain NBD‐labeled lipid.

FIGURE 6 Influence of various substances on the transport activity of Pdr5 in liposomes. (a) Oligomycin. (b) Clotrimazole. (c) Ketoconazole, and (d) Orthovanadate. The transport activity was normalized to the sample without addition of the substance and plotted against substance concentration. The error bars represent the mean ± SE of three independent measurements.

Similar to clotrimazole, the addition of ketoconazole resulted in a complete inhibition with a comparable Ki value of 9.4 ± 0.7 μM.

ATPase activity of reconstituted Pdr5 is also inhibited by oligomycin, ketoconazole, and clotrimazole (Figure 7). While complete inhibition of ATPase activity is evident for oligomycin with an IC50 of 0.64 ± 0.14 μM, ketoconazole and clotrimazole did not inhibit the ATPase activity quantitatively. Higher concentration of both compounds could not be used due to solubility problems. Additionally, a slight stimulation of ATPase activity in the presence of ketoconazole is visible. However, we believe it is not significant as it is on the order of 20% and likely within the accuracy of the assay. The incomplete inhibition of ATPase activity at high concentrations of ketoconazole and clotrimazole was sufficient to abolish the corresponding substrate transport (Figure 6) as Pdr5 is an uncoupled ABC transporter, for example, no strict coupling of ATPase activity and substrate translocation. Nevertheless, this result highlights that the energy of ATP is strictly required to translocate substrates in vitro.

FIGURE 7 Influence of various substances on the ATPase activity of Pdr5 in liposomes. (a) Oligomycin. (b) Ketoconazole. and (c) Clotrimazole. The ATPase activity was normalized to the sample without addition of the substance and plotted against substance concentration. The error bars represent the mean ± SE of three independent measurements.

3 DISCUSSION

As one of the most abundant ABC transporter in S. cerevisiae, Pdr5 was intensively studied in the last 30 years. Being a key player in the PDR network and conferring resistance against a large number of substances, resulted in a substantial interest in understanding the structure and function of this ABC transporter (Golin & Ambudkar, 2015). The structure of Pdr5 was elusive for numerous years since it was not possible to purify this ABC transporter in an active state. All attempts failed due to an inactivation of the protein caused by the detergent solubilization process. In 2019, a protocol describing the purification of Pdr5 in a homogenous and active state was published (Wagner et al., 2019). Moreover, it also showed the capability of Pdr5 in hydrolyzing all nucleotides and documented the first inhibition analysis of several substances in vitro. In light of this, the purification protocol paved the way for extensive structural (Harris et al., 2021) and functional investigations of Pdr5 (Wagner et al., 2019). While the analysis of the purified protein in detergent micelles provided the first insights into functional aspects, however, the analysis in the native environment was still missing.

Here, we describe a protocol for the reconstitution of purified Pdr5 in liposomes and conducted the first functional studies of Pdr5 in an isolated and native‐like environment. The provided reconstitution procedure yielded reproducibly proteoliposomes with an incorporation level of 58% (Figure 1). The amount of reconstituted Pdr5 into liposomes is consistent with what is known from literature for other proteins. For Cdr1from Candida albicans a protein recovery of 60% was determined (Shukla et al., 2007), while the amount for P‐gp reconstituted into liposomes was depending on the organism. Human P‐gp showed only a recovery of 10%, whereas the amount of reconstituted hamster P‐gp in nano‐scale liposomes yielded in 45%–55% (Park & Majd, 2018). A study on BmrA from Bacillus subtilis demonstrated, that the recovery rate after reconstitution was based on the lipid‐to‐protein ratio as well as the choice of detergent removal. Due to the use of dialysis in combination with bio‐beads for detergent removal the amount of reconstituted protein was up to 90%. When using solely bio‐beads, the recovery rate was limited to 50% due to the inhomogeneous detergent removal (Kunert et al., 2014).

Pdr5 was mostly incorporated unidirectional in an inside‐out orientation of 79% (Figure 1), allowing the hydrolysis and binding of ATP to the nucleotide‐binding sites. This is the optimal condition for addition of nucleotides externally to measure the ATPase activity of Pdr5 and validate its transport activity.

By employing an enzyme‐coupled assay that monitors the stoichiometric coupling of NTP hydrolysis to NADH oxidation, we were able to quantify the NTPase activity of Pdr5 reconstituted into liposomes (Figure 2). Our findings demonstrate that Pdr5 exhibits high basal ATPase activity, in line with observations conducted with plasma membrane vesicles and detergent‐solubilized protein (Decottignies et al., 1994; Ernst et al., 2008; Kolaczkowski et al., 1996; Wagner et al., 2019). For Pdr5 in detergent micelles the ATPase activity is limited to 209 nmol/(min*mg) at pH 9.5 (Wagner et al., 2019), whereas the v max for Pdr5 in plasma membrane vesicles ranges from 0.2 to 2.2 μmol/(min*mg) (Decottignies et al., 1994; Ernst et al., 2008; Golin et al., 2007; Gupta et al., 2014). For both systems it was shown, that the ATPase activity is highly depending on the buffer's pH (Gupta et al., 2014; Wagner et al., 2019). With a maximum velocity of 750 nmol/(min*mg), the ATPase activity of Pdr5 reconstituted into liposomes is higher compared to detergent micelles and lower than what is reported for plasma membrane vesicles. It should be noted that the ATPase activity for reconstituted Pdr5 was conducted in KPi buffer with a pH of 7.0. Considering the pH dependency of the activity, this value is quite comparable to that found in plasma membrane vesicles.

Earlier investigations already revealed the capability of Pdr5 to hydrolyze other nucleotides in addition to ATP (Decottignies et al., 1994; Golin et al., 2007; Wagner et al., 2019). Further, it was also shown for its homologue Cdr1 from Candida albicans, that it can hydrolyze other nucleotides (Nakamura et al., 2001). Our results support these observations and provide important insights into the substrate specificity and efficiencies of Pdr5 in hydrolyzing ATP, GTP, CTP, and UTP. Surprisingly, reconstituted Pdr5 exhibited the highest activity in the presence of CTP with a v max of 1202 nmol/(min*mg). Even though Pdr5 in detergent micelles also showed the highest velocity for CTP with 385 nmol/(min*mg), the overall hydrolysis efficiency for all nucleotides was at least 2.5‐fold lower compared to Pdr5 in liposomes (Wagner et al., 2019). For Pdr5 in plasma membrane vesicles, it was shown that the UTPase (Golin et al., 2007) activity was quite low indicating little to no physiological role of this pyrimidine nucleotide. CTPase activity has not been analyzed in plasma membrane vesicles so far. In liposomes, however, we could not observe a correlation between the nature of the nucleotide base and the maximum hydrolysis velocity (Table 1).

If the higher efficiency of reconstituted Pdr5 in hydrolyzing CTP is an artifact of the reconstitution process needs to be further validated. Nevertheless, taking the intracellular NTP concentrations into account, only ATP is present in sufficient amounts (Osorio et al., 2003). Despite this, in the case of cellular ATP limitations, this ABC transporter may be able to switch to other nucleotides as energy source and to fulfill its role in protecting the cell from xenobiotic compounds. The surprising high activity for CTP provides a foundation for further investigations into the mechanistics of nucleotide recognition and hydrolysis. The underlying structural differences in the nucleotide‐binding domains of Pdr5 while binding nucleotides other than ATP need to be studied in detail.

Moreover, in this study we could support early data suggesting the ability of Pdr5 to transport short‐chain NBD‐labeled lipids (Figure 4) (Decottignies et al., 1998). In our study, liposomes containing Pdr5 were labeled with 06:0–06:0 NBD‐PC and energized by addition of various nucleotides. In presence of ATP, we could observe a quantity of translocated NBD‐PC of around 60% after 100 min reaction time. Compared to this, the translocation of NBD‐phospholipids by P‐gp and Cdr1 was limited to 5%–10% depending on the headgroup, chain length and saturation level of the lipids (Romsicki & Sharom, 2001; Shukla et al., 2007). However, we like to stress that we are not proposing that Pdr5 acts as a lipid transporter. First, no lipid headgroup specificity was detected and second, long‐chain NBD lipids such as 12:0–06:0 or 16:0–06:0 NBD‐PC were not transported by Pdr5 reconstituted in liposomes. This clearly demonstrates that 06:0–06:0 NBD‐PC acts as an artificial reporter for substrate transport.

Furthermore, we also tested the ability of Pdr5 to translocate NBD‐PC in presence of CTP, UTP and GTP (Figure 5). Remarkably, compared to the other nucleotides, the amount of NBD‐PC transported in the presence of ATP was twice as much as for the other nucleotides. In a time course of 30 min the amount of transported NBD‐PC by using ATP reached 20%, whereas for the other nucleotides the amount was limited to 10%. These observations are contradictory to the results obtained for the NTPase activity demonstrating that the transport efficiency does not correlate with efficiency and rate of hydrolyzed nucleotides. For the NTPase activity the highest value was obtained in presence of CTP, which is not reflected in terms of phospholipid translocation.

Additionally, the translocation of short‐chain labeled NBD‐lipids is not specifically linked to the PC headgroup (Figure 5). We could show, that liposomes containing Pdr5 also transport NBD‐PA, NBD‐PE and NBD‐PG in comparable amounts with a slight, but hardly significant preference for NBD‐PG. Studies showing NBD‐labeled lipid transport by Cdr1 and P‐gp mainly used long‐chain labeled lipids with a small amount of transported lipids compared to this study (Romsicki & Sharom, 2001; Shukla et al., 2007; Smriti et al., 2002). In addition, these NBD‐lipid variants were not transported by Pdr5.

The addition of several substances for the transport assay revealed some important insights (Figure 6). Both oligomycin and orthovanadate are known to inhibit the ATPase activity of Pdr5. While oligomycin can also inhibit the F1FO ATP synthase and is widely used to inhibit the specific ATPase activity of PDR transporter in crude plasma membrane preparations (Lamping et al., 2007), orthovanadate inhibits ABC transporter by mimicking the transition state of the y‐phosphate of ATP and trapping the transporter in an outward‐facing conformation (Harris et al., 2021; Loo & Clarke, 2002).

Furthermore, the effect of the well‐known Pdr5 substrates ketoconazole and clotrimazole on the transport activity was determined. Both substrates fully inhibited the NBD‐labeled lipid transport with Ki values of 9.4 ± 0.7 μM for ketoconazole and 8.1 ± 0.8 μM for clotrimazole. Earlier investigations revealed that the ATPase activity of Pdr5 was affected by small amounts of ketoconazole and clotrimazole. Pdr5 in plasma membrane vesicles showed a Ki of 5.4 μM when exposed to ketoconazole and 2.5 μM when exposed to clotrimazole. From this perspective, it appears that the reduced NBD‐lipid transport activity might result from an inhibition of ATPase function. This assumption should be treated with caution, as it involves a comparison between data obtained from liposomes and plasma membrane vesicles. Even when comparing plasma membrane vesicles and Pdr5 in detergent micelles, a disparity between the Ki values in presence of ketoconazole was noticeable.

Consequently, it is crucial for upcoming research to investigate the ATPase activity of Pdr5 in liposomes in presence of the known substrates.

4 CONCLUSIONS

The reconstitution protocol allowed us to characterize Pdr5 in a well‐defined isolated and native‐like environment. We could demonstrate robust NTPase activity of this ABC transporter and present a transport assay, which can be used to further characterize various substances. Further structural analysis with different NTPs and substrates could provide us with details about nucleotide recognition and the correlation between NTP hydrolysis efficiency and utilization of the energy for translocation.

5 MATERIALS AND METHODS

5.1 Chemicals

All chemicals, if not stated otherwise, were obtained from Sigma‐Aldrich, Carl Roth, VWR or Thermo Fisher Scientific. POPE, POPC, POPS, 06:0–06:0 NBD‐PC, 06:0–06:0 NBD‐PA, 06:0–06:0 NBD‐PE, 06:0–06:0 NBD‐PG, 14:0–06:0 NBD‐PC and 16:0–06:0 NBD‐PC were purchased from Avanti Polar Lipids. Ergosterol, NADH and trans‐PCC‐α‐M were purchased from Fluka, GERBU Biotechnik and Glycon Biochemicals, respectively. Bio‐Beads SM‐2 beads were purchased from Bio‐Rad Laboratories and prepared as described by Geertsma et al. (Geertsma et al., 2008).

5.2 Protein expression and purification

Pdr5 was expressed and purified as described in Wagner et al. (Wagner et al., 2019) and is briefly summarized here.

Cells from S. cerevisiae strain YRE1001 (Ernst et al., 2008) were incubated at 30°C in YPD medium to a final OD600 of 3.5 to 4.0. The cells were harvested and disrupted using glass beads. Cell debris was removed by differential centrifugation at 4°C (twice at 1000×g for 5 min and once at 3000×g for 10 min). Membranes were collected by centrifugation at 20,000×g for 40 min. The pellet was resuspended in Buffer A (50 mM Tris–HCl pH 7.8, 50 mM NaCl and 10% (w/v) glycerol) and adjusted to 10 mg/ml total protein concentration.

Membrane proteins were solubilized with 1% (w/v) trans‐PCC‐α‐M at 4°C for 90 min under gentle stirring. Non‐solubilized material was removed by centrifugation at 170,000×g for 45 min at 4°C and the sample was loaded onto a 1 ml HiTrap Chelating column loaded with Zn2+ ions and equilibrated with low histidine buffer (50 mM Tris–HCl pH 7.8, 500 mM NaCl, 10% glycerol, 2.5 mM L‐histidine, 0.003% (w/v) trans‐PCC‐α‐M). The sample was washed and eluted stepwise using low and high histidine buffer (50 mM Tris–HCl pH 7.8, 500 mM NaCl, 10% glycerol, 100 mM L‐histidine, 0.003% (w/v) trans‐PCC‐α‐M). Fractions from the elution peak were pooled and concentrated using a Vivaspin™ Turbo 15 (50 kDa MWCO). The concentrated sample was purified via size exclusion chromatography (SEC) using a Superdex 200 Increase 10/300 GL column equilibrated with Buffer A containing 0.003% trans‐PCC‐α‐M.

5.3 Preparation of liposomes

The preparation of liposomes for the reconstitution of Pdr5 was performed according to Geertsma et al. (Geertsma et al., 2008). Stock solutions of POPC, POPE, POPS and ergosterol in chloroform were mixed in a 30:30:20:20 (w/w) ratio to a final amount of 40 mg lipids. Chloroform was removed using a rotary evaporator for ~45 min at 40°C to dry the lipids. The lipid film was suspended in 50 mM KPi pH 7.0 and adjusted to a total concentration of 20 mg/ml obtaining a homogeneous suspension. To create small unilamellar vesicles (SUVs), the suspension was sonicated in six cycles (5 s on/5 s off) on ice‐cold water. The suspension was flash‐frozen with liquid nitrogen and thawed at room temperature five times to generate large multilamellar vesicles (LMVs), which were stored at −80°C until used.

5.4 Reconstitution of purified Pdr5 into liposomes

Lipids were thawed at room temperature and extruded 11 times through a 0.4 μM polycarbonate filter to form large unilamellar vesicles (LUVs). 50 mM KPi pH 7.0 was added to dilute the LUVs to a final concentration of 4 mg/ml. Titration with 10% (w/v) Triton X‐100 was accomplished to destabilize the LUVs for the incorporation of protein. Subsequently, small amounts of Triton X‐100 were added and the absorption was monitored at 540 nm. The amount of detergent was increased until the absorption reached ~60% of the initial value before addition of detergent.

Purified Pdr5 was added to the destabilized liposomes in a protein‐to‐lipid ratio of 1:100 (w/w) and incubated for 15 min at room temperature under gentle agitation. 200 mg Bio‐Beads SM‐2 per 5 ml suspension were added and incubated at 4°C for 20 min under gentle agitation.

The Bio‐Beads were added two additional times and the suspension was incubated over night at 4°C under gentle agitation. On the next day, Bio‐Beads were again added and the suspension was incubated for further 90 min before the beads were removed via disposable Poly‐Prep chromatography columns. The proteoliposomes were stored at 8°C to a maximum of 1 week.

5.5 Efficiency of protein reconstitution and orientation of Pdr5 in proteoliposomes

The efficiency of protein reconstitution and the amount of reconstituted Pdr5 was determined via a quantitative SDS‐PAGE analysis. Prior to usage, the sample containing proteoliposomes was concentrated five‐fold by centrifugation at 220,000×g for 20 min at 4°C and resuspended in 50 mM KPi pH 7.0.

For analysis, 10 μl of purified protein with known concentrations as well as the proteoliposome sample of unknown concentration were loaded on a 7% SDS‐PAGE gel and separated by electrophoresis at 80 V. The gel was stained with Quick Coomassie Stain and the bands were analyzed via ImageJ software (Collins, 2007). The orientation of reconstituted protein was determined by a digestion experiment using trypsin. 40 μl of a 5‐fold concentrated proteoliposome sample was incubated with trypsin (2 μl at a concentration of 0.1 mg/ml) at 37°C for 4 h and stored at −20°C afterwards. As a control, 1% Triton X‐100 was added prior the addition of trypsin to solubilize the liposome. The stored samples were loaded onto a 7% SDS‐PAGE gel and analyzed as described before.

5.6 Enzyme‐coupled ATPase activity assay with proteoliposomes

ATPase activity of proteoliposomes containing Pdr5 was measured via an enzyme coupled assay. This assay is based on the stoichiometric oxidation of NADH to NAD+ due to the hydrolysis of ATP. The reaction was performed at 30°C in a 96‐well plate and monitored at 340 nm for 40 min. 50 mM KPi pH 7.0, 4 mM PEP pH 7.0, 0.6 mM NADH, 5 mM MgCl2, 13 units of pyruvate kinase and 15 units of lactate dehydrogenase were added into a well with a final volume of 170 μl. Directly before the measurement, 20 μl of proteoliposomes containing Pdr5 were added and the reaction was started by the addition of 10 μl of 100 mM ATP. The background measurement was performed by the addition of 50 mM KPi pH 7.0 instead of proteoliposomes and without ATP. For the NTP kinetic measurements, various concentrations of ATP, GTP, CTP or UTP ranging from 0 to 2 mM final concentration were added. The background measurements were carried out by the addition of 50 mM KPi pH 7.0 instead of proteoliposomes and with various concentrations of the NTPs.

For inhibitory measurements, 2 μl of the compounds (dissolved in an appropriate solvent) were added to the reaction mix in the wells and incubated for 10 min before proteoliposomes were added and the reaction was started with ATP.

5.7 NBD‐PC translocation in liposomes containing Pdr5

The transport activity of proteoliposomes containing Pdr5 was measured via a NBD‐labeled lipid translocation assay. For incorporation of the NBD‐labeled lipids into the outer leaflet of the liposomes, 50 μl proteoliposomes were incubated with 5.5 μl of 06:0–06:0 NBD‐PC (final concentration 5 μM) at 25°C for 20 min. To initiate the lipid translocation, 4 mM MgATP was added and the sample was incubated at 30°C. The reaction was terminated after certain time points by the addition of 2 mM sodium orthovanadate. The sample was kept on ice and analyzed via fluorescence quenching using an excitation wavelength of 468 nm (slit 2 nm) and an emission wavelength of 540 nm (slit 5 nm). 950 μl of KPi pH 7.0 was transferred to a 1 ml quartz cuvette and the fluorescence emission was recorded at 25°C. After the baseline was stable for ~100 s, 50 μl of the sample was added. Fluorescence quenching of NBD‐labeled lipids was performed by addition of 10 μl 100 mM sodium dithionite (final concentration of 1 mM) after ~100 s. Finally, after another time period of ~100 s liposomes were permeabilized by addition of 100 μl of 10% (w/v) Triton X‐100 (final concentration of 1%). The fluorescence measurement was continued for at least 50 s. Prolonged incubation times didn't lead to a significant different result. Control measurements were performed using 4 mM MgAMP instead of MgATP. The percentage of NBD‐lipid transport is substracted from the value in the presence of MgATP to obtain the value of ATP‐dependent NBD‐lipid transport. Additional experiments were performed with the corresponding concentration of MgGTP, MgCTP and MgUTP with various reaction times.

For testing the lipids specificity, the incorporation was carried out either with 06:0–06:0 NBD‐PC, 06:0–06:0 NBD‐PA, 06:0–06:0 NBD‐PE or 06:0–06:0 NBD‐PG for 20 min at 25°C. The transport assay was initiated with 4 mM MgATP and performed as described above.

For the inhibitory measurements, 2 μl of the compounds (dissolved in an appropriate solvent) were added to the proteoliposome sample labeled with NBD‐PC and incubated for 5 min before 4 mM MgATP was added to start the reaction. The reaction was terminated after 30 min incubation at 30°C.

The percentage of translocated NBD‐labeled lipid in proteoliposomes was calculated using Equation (1): (1) %NBD−labeled lipid in inner leaflet=FT−FD/FT−F0×100

where F T is the total fluorescence of the sample, FD is the fluorescence after addition of sodium dithionite and F0 is the fluorescence after addition of Triton X‐100.

AUTHOR CONTRIBUTIONS

Stefanie L. Gala Marti: Investigation; writing – original draft; methodology; formal analysis; data curation. Manuel Wagner: Investigation; methodology. Lea‐Marie Nentwig: Investigation; validation; methodology; formal analysis. Sander H. J. Smits: Writing – review and editing; supervision. Lutz Schmitt: Conceptualization; resources; supervision; formal analysis; writing – review and editing; project administration.

ACKNOWLEDGMENTS

We thank all current and former members of the Institute of Biochemistry for fruitful discussions. We are indebted to Prof. Bert Poolman, University of Groningen, for support during the early stages of the project. Research on Pdr5 was funded by the Deutsche Forschungsgemeinschaft (DFG, grant Schm1279/17‐1 to Lutz Schmitt). The Center for Structural Studies is funded by Deutsche Forschungsgemeinschaft (DFG, grant 417919780 to Sander H. J. Smits). Open Access funding enabled and organized by Projekt DEAL.
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REFERENCES

Alhumaidi M , Nentwig L‐M , Rahman H , Schmitt L , Rudrow A , Harris A , et al. Residues forming the gating regions of asymmetric multidrug transporter pdr5 also play roles in conformational switching and protein folding. J Biol Chem. 2022;298 (12 ):102689.36370844
Choi CC , Ford RC . Atp binding cassette importers in eukaryotic organisms. Biol Rev Camb Philos Soc. 2021;96 (4 ):1318–1330.33655617
Collins TJ . Imagej for microscopy. Biotechniques. 2007;43 (S1 ):S25–S30.
Decottignies A , Grant AM , Nichols JW , de Wet H , McIntosh DB , Goffeau A . Atpase and multidrug transport activities of the overexpressed yeast abc protein yor1p. J Biol Chem. 1998;273 (20 ):12612–12622.9575223
Decottignies A , Kolaczkowski M , Balzi E , Goffeau A . Solubilization and characterization of the overexpressed pdr5 multidrug resistance nucleotide triphosphatase of yeast. J Biol Chem. 1994;269 (17 ):12797–12803.8175692
Ernst R , Klemm R , Schmitt L , Kuchler K . Yeast atp‐binding cassette transporters: cellular cleaning pumps. Methods Enzymol. 2005;400:460–484.
Ernst R , Kueppers P , Klein CM , Schwarzmueller T , Kuchler K , Schmitt L . A mutation of the h‐loop selectively affects rhodamine transport by the yeast multidrug abc transporter pdr5. Proc Natl Acad Sci U S A. 2008;105 (13 ):5069–5074.18356296
Geertsma ER , Nik Mahmood NA , Schuurman‐Wolters GK , Poolman B . Membrane reconstitution of abc transporters and assays of translocator function. Nat Protoc. 2008;3 (2 ):256–266.18274528
Golin J , Ambudkar SV . The multidrug transporter pdr5 on the 25th anniversary of its discovery: an important model for the study of asymmetric abc transporters. Biochem J. 2015;467 :353–363.25886173
Golin J , Kon ZN , Wu C‐P , Martello J , Hanson L , Supernavage S , et al. Complete inhibition of the pdr5p multidrug efflux pump atpase activity by its transport substrate clotrimazole suggests that gtp as well as atp may be used as an energy source. Biochemistry. 2007;46 (45 ):13109–13119.17956128
Gottesman MM , Fojo T , Bates SE . Multidrug resistance in cancer: role of atp–dependent transporters. Nat Rev Cancer. 2002;2 (1 ):48–58.11902585
Gupta RP , Kueppers P , Hanekop N , Schmitt L . Generating symmetry in the asymmetric atp‐binding cassette (abc) transporter pdr5 from Saccharomyces cerevisiae . J Biol Chem. 2014;289 (22 ):15272–15279.24733388
Harris A , Wagner M , Du D , Raschka S , Nentwig LM , Gohlke H , et al. Structure and efflux mechanism of the yeast pleiotropic drug resistance transporter pdr5. Nat Commun. 2021;12 (1 ):5254.34489436
Holland IB , Cole SP , Kuchler K , Higgins CF . Abc proteins: from bacteria to man. Amsterdam: Elsevier; 2003.
Kolaczkowski M , van der Rest M , Cybularz‐Kolaczkowska A , Soumillion JP , Konings WN , Goffeau A . Anticancer drugs, ionophoric peptides, and steroids as substrates of the yeast multidrug transporter pdr5p. J Biol Chem. 1996;271 (49 ):31543–31548.8940170
Kunert B , Gardiennet C , Lacabanne D , Calles‐Garcia D , Falson P , Jault J‐M , et al. Efficient and stable reconstitution of the abc transporter bmra for solid‐state nmr studies. Front Mol Biosci. 2014;1 :5.25988146
Lage H . Abc‐transporters: implications on drug resistance from microorganisms to human cancers. Int J Antimicrob Agents. 2003;22 (3 ):188–199.13678820
Lamping E , Baret PV , Holmes AR , Monk BC , Goffeau A , Cannon RD . Fungal pdr transporters: phylogeny, topology, motifs and function. Fungal Genet Biol. 2010;47 (2 ):127–142.19857594
Lamping E , Monk BC , Niimi K , Holmes AR , Tsao S , Tanabe K , et al. Characterization of three classes of membrane proteins involved in fungal azole resistance by functional hyperexpression in Saccharomyces cerevisiae . Eukaryot Cell. 2007;6 (7 ):1150–1165.17513564
Loo TW , Clarke DM . Vanadate trapping of nucleotide at the atp‐binding sites of human multidrug resistance p‐glycoprotein exposes different residues to the drug‐binding site. Proc Natl Acad Sci U S A. 2002;99 (6 ):3511–3516.11891276
Nakamura K , Niimi M , Niimi K , Holmes AR , Yates JE , Decottignies A , et al. Functional expression of candida albicans drug efflux pump cdr1p in a Saccharomyces cerevisiae strain deficient in membrane transporters. Antimicrob Agents Chemother. 2001;45 (12 ):3366–3374.11709310
Osorio H , Carvalho E , del Valle M , Günther Sillero MA , Moradas‐Ferreira P , Sillero A . H2O2, but not menadione, provokes a decrease in the atp and an increase in the inosine levels in Saccharomyces cerevisiae . Eur J Biochem. 2003;270 (7 ):1578–1589.12654013
Oswald C , Holland IB , Schmitt L . The motor domains of abc‐transporters. What can structures tell us? Naunyn Schmiedebergs Arch Pharmacol. 2006;372 (6 ):385–399.16541253
Park S , Majd S . Reconstitution and functional studies of hamster p‐glycoprotein in giant liposomes. PLoS One. 2018;13 (6 ):e0199279.29912971
Romsicki Y , Sharom FJ . Phospholipid flippase activity of the reconstituted p‐glycoprotein multidrug transporter. Biochemistry. 2001;40 (23 ):6937–6947.11389609
Schmitt L , Tampe R . Structure and mechanism of abc transporters. Curr Opin Struct Biol. 2002;12 (6 ):754–760.12504680
Shen HH , Lithgow T , Martin L . Reconstitution of membrane proteins into model membranes: seeking better ways to retain protein activities. Int J Mol Sci. 2013;14 (1 ):1589–1607.23344058
Shukla S , Rai V , Saini P , Banerjee D , Menon AK , Prasad R . Candida drug resistance protein 1, a major multidrug atp binding cassette transporter of candida albicans, translocates fluorescent phospholipids in a reconstituted system. Biochemistry. 2007;46 (43 ):12081–12090.17924650
Smriti KS , Dixit BL , Gupta CM , Milewski S , Prasad R . Abc transporters cdr1p, cdr2p and cdr3p of a human pathogen candida albicans are general phospholipid translocators. Yeast. 2002;19 (4 ):303–318.11870854
Stockner T , Gradisch R , Schmitt L . The role of the degenerate nucleotide binding site in type i abc exporters. FEBS Lett. 2020;594 (23 ):3815–3838.33179257
van't Klooster JS , Cheng TY , Sikkema HR , Jeucken A , Moody DB , Poolman B . Membrane lipid requirements of the lysine transporter lyp1 from Saccharomyces cerevisiae . J Mol Biol. 2020;432 (14 ):4023–4031.32413406
Wagner M , Smits SH , Schmitt L . In vitro ntpase activity of highly purified pdr5, a major yeast abc multidrug transporter. Sci Rep. 2019;9 (1 ):7761.31123301
