Transporters are transmembrane proteins that mediate the selective translocation of solutes across biological membranes. Recently, we have shown that specific interactions with plasma membrane phospholipids are essential for the formation and/or stability of functional dimers of the purine transporter UapA, a prototypic eukaryotic member of the ubiquitous nucleobase ascorbate transporter (NAT) family. Here, we provide strong evidence that distinct interactions of UapA with membrane lipids are essential for ab initio formation of functional dimers in the ER, or ER exit and further subcellular trafficking. Through genetic screens, we identify mutations that restore defects in dimer formation and/or trafficking. Suppressors of defective dimerization restore ab initio formation of UapA dimers in the ER. Most of these suppressors are located in the movable core domain, but also in the core-dimerization interface and in residues of the dimerization domain exposed to lipids. Molecular dynamics suggest that the majority of suppressors stabilize interhelical interactions in the core domain and thus assist the formation of functional UapA dimers. Among suppressors restoring dimerization, a specific mutation, T401P, was also isolated independently as a suppressor restoring trafficking, suggesting that stabilization of the core domain restores function by sustaining structural defects caused by the abolishment of essential interactions with specific lipids. Importantly, the introduction of mutations topologically equivalent to T401P into a rat homolog of UapA, namely rSNBT1, permitted the functional expression of a mammalian NAT in Aspergillus nidulans Thus, our results provide a potential route for the functional expression and manipulation of mammalian transporters in the model Aspergillus system.

中文翻译：

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嘌呤转运蛋白中的特定残基对于二聚化、ER 退出和功能至关重要。

转运蛋白是介导溶质跨生物膜选择性易位的跨膜蛋白。最近，我们发现与质膜磷脂的特异性相互作用对于嘌呤转运蛋白 UapA 功能二聚体的形成和/或稳定性至关重要，UapA 是普遍存在的核碱基抗坏血酸转运蛋白 (NAT) 家族的原型真核成员。在这里，我们提供了强有力的证据，证明 UapA 与膜脂的独特相互作用对于ER 或 ER 出口中功能二聚体的从头形成以及进一步的亚细胞运输至关重要。通过基因筛选，我们识别出可恢复二聚体形成和/或运输缺陷的突变。有缺陷的二聚化抑制剂可恢复ER 中 UapA 二聚体的从头形成。大多数这些抑制因子位于可移动核心结构域中，但也位于核心二聚化界面和暴露于脂质的二聚化结构域的残基中。分子动力学表明，大多数抑制子稳定核心结构域中的螺旋间相互作用，从而有助于功能性 UapA 二聚体的形成。在恢复二聚化的抑制子中，一个特定的突变 T401P 也被独立分离为恢复运输的抑制子，这表明核心结构域的稳定通过维持由消除与特定脂质的基本相互作用引起的结构缺陷来恢复功能。重要的是，将拓扑等同于 T401P 的突变引入 UapA 的大鼠同源物（即 rSNBT1）中，允许哺乳动物 NAT 在构巢曲霉中功能性表达。因此，我们的结果为哺乳动物转运蛋白在构巢曲霉中的功能性表达和操作提供了潜在的途径。模型曲霉菌系统。