{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,7,30]],"date-time":"2025-07-30T10:06:27Z","timestamp":1753869987649,"version":"3.41.2"},"reference-count":32,"publisher":"Wiley","issue":"10","license":[{"start":{"date-parts":[[2024,9,23]],"date-time":"2024-09-23T00:00:00Z","timestamp":1727049600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["Schm1279\/17\u20101","417919780"],"award-info":[{"award-number":["Schm1279\/17\u20101","417919780"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Protein Science"],"published-print":{"date-parts":[[2024,10]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Pdr5 is the most abundant ABC transporter in <jats:italic>Saccharomyces cerevisiae<\/jats:italic> 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 <jats:italic>Candida albicans<\/jats:italic>. In the past 30\u2009years, 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\u2010like environment in an almost unidirectional inside\u2010out orientation is described. We demonstrate that reconstituted Pdr5 is capable of translocating short\u2010chain 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.<\/jats:p>","DOI":"10.1002\/pro.5181","type":"journal-article","created":{"date-parts":[[2024,9,23]],"date-time":"2024-09-23T15:44:37Z","timestamp":1727106277000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["An in vitro set\u2010up to study Pdr5\u2010mediated substrate translocation"],"prefix":"10.1002","volume":"33","author":[{"given":"Stefanie L.","family":"Gala Marti","sequence":"first","affiliation":[{"name":"Institute of Biochemistry Heinrich Heine University D\u00fcsseldorf  D\u00fcsseldorf Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Manuel","family":"Wagner","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry Heinrich Heine University D\u00fcsseldorf  D\u00fcsseldorf Germany"},{"name":"OQEMA GmbH  M\u00f6nchengladbach Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lea\u2010Marie","family":"Nentwig","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry Heinrich Heine University D\u00fcsseldorf  D\u00fcsseldorf Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sander H. J.","family":"Smits","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry Heinrich Heine University D\u00fcsseldorf  D\u00fcsseldorf Germany"},{"name":"Center for Structural Studies Heinrich Heine University D\u00fcsseldorf  D\u00fcsseldorf Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1167-9819","authenticated-orcid":false,"given":"Lutz","family":"Schmitt","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry Heinrich Heine University D\u00fcsseldorf  D\u00fcsseldorf Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2024,9,23]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbc.2022.102689"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1111\/brv.12702"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.2144\/000112517"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.273.20.12612"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(18)99946-1"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0076-6879(05)00026-1"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0800191105"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2007.519"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1042\/BJ20150042"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi701414f"},{"key":"e_1_2_8_12_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrc706"},{"key":"e_1_2_8_13_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M114.553065"},{"key":"e_1_2_8_14_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-25574-8"},{"volume-title":"Abc proteins: from bacteria to man","year":"2003","author":"Holland IB","key":"e_1_2_8_15_1"},{"key":"e_1_2_8_16_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.271.49.31543"},{"key":"e_1_2_8_17_1","doi-asserted-by":"publisher","DOI":"10.3389\/fmolb.2014.00005"},{"key":"e_1_2_8_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0924-8579(03)00203-6"},{"key":"e_1_2_8_19_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.fgb.2009.10.007"},{"key":"e_1_2_8_20_1","doi-asserted-by":"publisher","DOI":"10.1128\/EC.00091-07"},{"key":"e_1_2_8_21_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.022049799"},{"key":"e_1_2_8_22_1","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.45.12.3366-3374.2001"},{"key":"e_1_2_8_23_1","doi-asserted-by":"publisher","DOI":"10.1046\/j.1432-1033.2003.03529.x"},{"key":"e_1_2_8_24_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00210-005-0031-4"},{"key":"e_1_2_8_25_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0199279"},{"key":"e_1_2_8_26_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi0024456"},{"key":"e_1_2_8_27_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0959-440X(02)00399-8"},{"key":"e_1_2_8_28_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms14011589"},{"key":"e_1_2_8_29_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi700453e"},{"key":"e_1_2_8_30_1","doi-asserted-by":"publisher","DOI":"10.1002\/yea.818"},{"key":"e_1_2_8_31_1","doi-asserted-by":"publisher","DOI":"10.1002\/1873-3468.13997"},{"key":"e_1_2_8_32_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2020.04.029"},{"key":"e_1_2_8_33_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-019-44327-8"}],"container-title":["Protein Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/pro.5181","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,9,23]],"date-time":"2024-09-23T15:44:39Z","timestamp":1727106279000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/pro.5181"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,9,23]]},"references-count":32,"journal-issue":{"issue":"10","published-print":{"date-parts":[[2024,10]]}},"alternative-id":["10.1002\/pro.5181"],"URL":"https:\/\/doi.org\/10.1002\/pro.5181","archive":["Portico"],"relation":{},"ISSN":["0961-8368","1469-896X"],"issn-type":[{"type":"print","value":"0961-8368"},{"type":"electronic","value":"1469-896X"}],"subject":[],"published":{"date-parts":[[2024,9,23]]},"assertion":[{"value":"2024-05-14","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2024-09-06","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2024-09-23","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e5181"}}