In vitro NTPase activity of highly purified Pdr5, a major yeast ABC multidrug transporter

Abstract

The ABC transporter Pdr5 of S . cerevisiae is a key player of the PDR network that works as a first line of defense against a wide range of xenobiotic compounds. As the first discovered member of the family of asymmetric PDR ABC transporters, extensive studies have been carried out to elucidate the molecular mechanism of drug efflux and the details of the catalytic cycle. Pdr5 turned out to be an excellent model system to study functional and structural characteristics of asymmetric, uncoupled ABC transporters. However, to date studies have been limited to in vivo or plasma membrane systems, as it was not possible to isolate Pdr5 in a functional state. Here, we describe the solubilization and purification of Pdr5 to homogeneity in a functional state as confirmed by in vitro assays. The ATPase deficient Pdr5 E1036Q mutant was used as a control and proves that detergent-purified wild-type Pdr5 is functional resembling in its activity the one in its physiological environment. Finally, we show that the isolated active Pdr5 is monomeric in solution. Taken together, our results described in this study will enable a variety of functional investigations on Pdr5 required to determine molecular mechanism of this asymmetric ABC transporter.

Functional Significance of Conserved Cysteines in the Extracellular Loops of the ATP Binding Cassette Transporter Pdr11p

“…In line with this notion, all purified cysteine mutants exhibited a stable basal ATPase activity, ruling out a direct effect on the functionality of the transporter. This basal activity is within the range of values reported for a number of other purified eukaryotic ABC transporters [ 26 , 27 , 28 , 29 , 30 ]. Two purified detergent-solubilized cysteine mutants (C1330S and C1333S) exhibited ~2-fold increased basal ATP activity.…”

Characterization of the nucleotide-binding domain NsrF from the BceAB-type ABC-transporter NsrFP from the human pathogen Streptococcus agalactiae

“…With regards to the substrate affinity represented by the k half value, a minimum of 0.41 ± 0.05 mM was reached using ATP as a substrate, which signifies ATP as the most favoured of all four tested NTPs for SaNsrF WT . Hence, ATP has the highest affinity to SaNsrF WT compared to the other examined NTPs, which corresponds with the physiological appearance in vivo of each NTP ([ATP] > [GTP] > [UTP] > [CTP]), which underlines that ATP is the preferred substrate for the protein 32,[66][67][68] . Considering the physiology of purine (ATP, GTP) and pyrimidine (UTP, CTP) nucleotides, we concluded that the involved aromatic ring systems play a major role concerning the substrate affinity and stability of the protein-substrate-complex.…”

“…An interaction between the NBD and the nucleotide is supposed to occur by π–π-stacking between the aromatic ring system of the nucleotide and an aromatic residue of the protein (F or Y). Hence, no preference towards any nucleotide-triphosphate (NTP) has been assumed 24 , as also observed for example for yeast PDR5 32 . The hydrolysis of ATP is coupled to the presence of a cofactor, almost exclusively Mg 2+ , which is coordinated by the Walker B motif.…”

Transcription factors and ABC transporters: from pleiotropic drug resistance to cellular signaling in yeast

“…[32] demonstrated that the regulatory domains of both Pdr1 and Pdr3 bind the antifungal ketoconazole at low micromolar affinity, leading to the speculation that ligand binding may increase transcriptional activity. Characterization of Pdr1 mutants has also proven useful in the yeast membrane biology field as pdr1‐3 has proven an effective tool for overexpression, and hence biochemical study, of Pdr5 and other membrane proteins [33,34].…”