研究 Pdr5 介导的底物转运的体外装置。

IF 4.5 3区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Protein Science Pub Date : 2024-10-01 DOI:10.1002/pro.5181
Stefanie L Gala Marti, Manuel Wagner, Lea-Marie Nentwig, Sander H J Smits, Lutz Schmitt
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引用次数: 0

摘要

Pdr5 是酿酒酵母(Saccharomyces cerevisiae)中最丰富的 ABC 转运体,在多效性耐药性(PDR)网络中发挥着重要作用,它能主动阻止大量结构不相关的化合物进入细胞。由于 Pdr5 的一个核苷酸结合位点(NBS)具有高度不对称性,因此它是不对称 ABC 转运体的完美模型系统,例如它与医学相关的白念珠菌同源物 Cdr1。在过去的 30 年中,人们对这种 ABC 转运体在体内和质膜囊泡中的情况进行了深入研究。然而,由于无法在合成膜系统中分离和重组 Pdr5 并保持其活性,这些研究受到了限制。本文描述了 Pdr5 在类似原生环境中几乎单向内向外的功能重组。我们证明重组的 Pdr5 能够将短链荧光 NBD 脂质从蛋白脂质体的外叶转运到内叶。此外,该转运体还揭示了其在重组系统中利用其他核苷酸完成底物转运的能力。此外,我们还估算了重组 Pdr5 的 NTP 酶活性,并确定了 ATP、GTP、CTP 和 UTP 的动力学参数。
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An in vitro set-up to study Pdr5-mediated substrate translocation.

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.

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来源期刊
Protein Science
Protein Science 生物-生化与分子生物学
CiteScore
12.40
自引率
1.20%
发文量
246
审稿时长
1 months
期刊介绍: Protein Science, the flagship journal of The Protein Society, is a publication that focuses on advancing fundamental knowledge in the field of protein molecules. The journal welcomes original reports and review articles that contribute to our understanding of protein function, structure, folding, design, and evolution. Additionally, Protein Science encourages papers that explore the applications of protein science in various areas such as therapeutics, protein-based biomaterials, bionanotechnology, synthetic biology, and bioelectronics. The journal accepts manuscript submissions in any suitable format for review, with the requirement of converting the manuscript to journal-style format only upon acceptance for publication. Protein Science is indexed and abstracted in numerous databases, including the Agricultural & Environmental Science Database (ProQuest), Biological Science Database (ProQuest), CAS: Chemical Abstracts Service (ACS), Embase (Elsevier), Health & Medical Collection (ProQuest), Health Research Premium Collection (ProQuest), Materials Science & Engineering Database (ProQuest), MEDLINE/PubMed (NLM), Natural Science Collection (ProQuest), and SciTech Premium Collection (ProQuest).
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