Escobar Adrian, Styrpejko Daniel J, Ali Saima, Cuajungco Math P
Department of Biological Science, USA.
Center for Applied Biotechnology Studies, California State University Fullerton, CA, 92831, USA.
Biochem Biophys Rep. 2022 Oct 1;32:101362. doi: 10.1016/j.bbrep.2022.101362. eCollection 2022 Dec.
Recently, we reported that TMEM163 is a zinc efflux transporter that likely belongs to the mammalian solute carrier 30 (Slc30/ZnT) subfamily of the cation diffusion facilitator (CDF) protein superfamily. We hypothesized that human TMEM163 forms functional heterodimers with certain ZNT proteins based on their overlapping subcellular localization with TMEM163 and previous reports that some ZNT monomers interact with each other. In this study, we heterologously expressed individual constructs with a unique peptide tag containing TMEM163, ZNT1, ZNT2, ZNT3, and ZNT4 (negative control) or co-expressed TMEM163 with each ZNT in cultured cells for co-immunoprecipitation (co-IP) experiments. We also co-expressed TMEM163 with two different peptide tags as a positive co-IP control. Western blot analyses revealed that TMEM163 dimerizes with itself but that it also heterodimerizes with ZNT1, ZNT2, ZNT3, and ZNT4 proteins. Confocal microscopy revealed that TMEM163 and ZNT proteins partially co-localize in cells, suggesting that they exist as homodimers and heterodimers in their respective subcellular sites. Functional zinc flux assays using Fluozin-3 and Newport Green dyes show that TMEM163/ZNT heterodimers exhibit similar efflux function as TMEM163 homodimers. Cell surface biotinylation revealed that the plasma membrane localization of TMEM163 is not markedly influenced by ZNT co-expression. Overall, our results show that the interaction between TMEM163 and distinct ZNT proteins is physiologically relevant and that their heterodimerization may serve to increase the functional diversity of zinc effluxers within specific tissues or cell types.
最近，我们报道了TMEM163是一种锌外流转运蛋白，可能属于阳离子扩散促进剂（CDF）蛋白超家族的哺乳动物溶质载体30（Slc30/ZnT）亚家族。基于它们与TMEM163重叠的亚细胞定位以及先前关于一些ZnT单体相互作用的报道，我们推测人类TMEM163与某些ZnT蛋白形成功能性异二聚体。在本研究中，我们在培养细胞中异源表达了含有TMEM163、ZnT1、ZnT2、ZnT3和ZnT4（阴性对照）的带有独特肽标签的单个构建体，或者将TMEM163与每种ZnT共表达以进行免疫共沉淀（co-IP）实验。我们还将TMEM163与两种不同的肽标签共表达作为阳性co-IP对照。蛋白质印迹分析显示，TMEM163能与自身形成二聚体，但也能与ZnT1、ZnT2、ZnT3和ZnT4蛋白形成异二聚体。共聚焦显微镜检查显示，TMEM163和ZnT蛋白在细胞中部分共定位，表明它们在各自的亚细胞位点以同二聚体和异二聚体形式存在。使用Fluozin-3和Newport Green染料进行的功能性锌通量测定表明，TMEM163/ZnT异二聚体与TMEM163同二聚体具有相似的外流功能。细胞表面生物素化显示，ZnT共表达对TMEM163的质膜定位没有明显影响。总体而言，我们的结果表明，TMEM163与不同ZnT蛋白之间的相互作用具有生理相关性，它们的异二聚化可能有助于增加特定组织或细胞类型中锌外流蛋白的功能多样性。
