Stanchev Lyubomir Dimitrov, Marek Magdalena, Xian Feng, Klöhn Mara, Silvestro Daniele, Dittmar Gunnar, López-Marqués Rosa Laura, Günther Pomorski Thomas
Department of Molecular Biochemistry, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, 44801 Bochum, Germany.
Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark.
J Fungi (Basel). 2020 Dec 22;7(1):2. doi: 10.3390/jof7010002.
The pleiotropic drug resistance (PDR) transporter Pdr11p is expressed under anaerobic growth conditions at the plasma membrane of the yeast , where it facilitates the uptake of exogenous sterols. Members of the fungal PDR family contain six conserved cysteines in their extracellular loops (ECL). For the functional analysis of these cysteine residues in Pdr11p, we generated a series of single cysteine-to-serine mutants. All mutant proteins expressed well and displayed robust ATPase activity upon purification. Mass-spectrometry analysis identified two cysteine residues (C582 and C603) in ECL3 forming a disulfide bond. Further characterization by cell-based assays showed that all mutants are compromised in facilitating sterol uptake, protein stability, and trafficking to the plasma membrane. Our data highlight the fundamental importance of all six extracellular cysteine residues for the functional integrity of Pdr11p and provide new structural insights into the PDR family of transporters.
多药耐药性（PDR）转运蛋白Pdr11p在酵母质膜的厌氧生长条件下表达，在那里它促进外源性固醇的摄取。真菌PDR家族成员在其细胞外环（ECL）中含有六个保守的半胱氨酸。为了对Pdr11p中的这些半胱氨酸残基进行功能分析，我们构建了一系列单半胱氨酸到丝氨酸的突变体。所有突变蛋白表达良好，纯化后显示出强大的ATP酶活性。质谱分析确定了ECL3中的两个半胱氨酸残基（C582和C603）形成了一个二硫键。通过基于细胞的试验进一步表征表明，所有突变体在促进固醇摄取、蛋白质稳定性和向质膜运输方面都受到损害。我们的数据突出了所有六个细胞外半胱氨酸残基对Pdr11p功能完整性的根本重要性，并为PDR转运蛋白家族提供了新的结构见解。
Zotero 插件
