Year: 2024
Type: article
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.
Source: Protein Science
DOI: 10.1002/pro.5181
Authors: Stefanie L. Gala Marti, Manuel Wagner, Lea‐Marie Nentwig, Sander H. J. Smits, Lutz Schmitt
Institution: Heinrich Heine University Düsseldorf
Language: English
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: hybrid
Estimated APC paid: $4,270
OpenAlex ID: W4402745491