Interaction proteomics of the human solute carrier (SLC) transporter superfamily

Solute carrier (SLC) transporters form a major superfamily which transport a wide range of substrates across cellular and organellar membranes. The superfamily consists of about 450 members and includes passive transporters, antiporters and symporters. To ensure the homeostatic regulation of metabolism and energetic household, transporters need to act in concert and be linked to the regulatory processes and pathways of cells. While there is ample evidence that regulation of transporter activity requires protein interactions, a comprehensive interactome of the whole human transproteome has not been described. We report here the first characterisation of the interactome of SLC transporters. To this aim, we applied an affinity purification combined with mass spectrometry (AP-MS) protocol tailored to transmembrane proteins to map the protein interactome for 400 solute carriers. The generated interactome covers thousands of novel protein-protein interactions, therefore providing a basis for understanding the molecular regulation of this protein superfamily. The SLC-interactome was dissected by clustering analysis, including SLC interactome-profile similarity-based clustering. The clustering revealed the association of SLCs with protein folding and trafficking. Thus, a selected set of interaction partners, associated with the proteostatic and trafficking regulation of SLCs, were selected for validation. We employed a high-throughput protein stability assay in combination with genetic perturbation of these selected interaction partners, to identify interactors which influence the abundance levels of SLCs or their subcellular locations. For selected interactions we performed transporter activity assays to show a direct modulation of transporter activity upon perturbation of protein interactions. Our work provides thus important insight into the complex molecular network of SLCs.

溶质载体（Solute carrier, SLC）转运蛋白是一类主要的跨膜转运蛋白超家族，负责介导各类底物跨细胞膜与细胞器膜的转运。该超家族包含约450个成员，涵盖被动转运蛋白、反向转运体与同向转运体。为维持代谢与能量稳态的调控平衡，转运蛋白需协同发挥作用，并与细胞内的调控过程及信号通路相耦联。尽管已有充分证据表明，转运蛋白活性的调控依赖于蛋白质相互作用的介导，但目前尚未有针对人类全转运蛋白质组的完整相互作用组的相关报道。

本研究首次对SLC转运蛋白的相互作用组进行了系统表征。为此，我们采用了针对跨膜蛋白优化的亲和纯化联合质谱（affinity purification combined with mass spectrometry, AP-MS）实验方案，对400种溶质载体蛋白的蛋白质相互作用组进行了图谱绘制。本次构建的相互作用组涵盖了数千种全新的蛋白质-蛋白质相互作用，为解析该蛋白超家族的分子调控机制提供了重要基础。

我们通过聚类分析对SLC相互作用组进行了解构，其中包括基于SLC相互作用组谱相似性的聚类方法。该聚类分析结果揭示了SLC转运蛋白与蛋白质折叠及囊泡转运过程之间的关联。因此，我们筛选出一批与SLC蛋白质稳态及转运调控相关的相互作用伴侣蛋白，用于后续验证实验。我们采用高通量蛋白质稳定性检测技术，结合针对上述筛选得到的相互作用伴侣的遗传扰动实验，以鉴定能够影响SLC蛋白丰度或其亚细胞定位的相互作用因子。针对部分筛选出的相互作用对，我们开展了转运蛋白活性检测实验，证实了蛋白质相互作用扰动可直接调控转运蛋白的活性。综上，本研究为解析SLC转运蛋白的复杂分子调控网络提供了重要见解。