In vitro NTPase activity of highly purified Pdr5, a major yeast ABC multidrug transporter.
Sci Rep
; 9(1): 7761, 2019 05 23.
Article
em En
| MEDLINE
| ID: mdl-31123301
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Transportadores de Cassetes de Ligação de ATP
/
Proteínas de Saccharomyces cerevisiae
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Nucleosídeo-Trifosfatase
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
Sci Rep
Ano de publicação:
2019
Tipo de documento:
Article