Transmembrane 163 (TMEM163) protein interacts with specific mammalian SLC30 zinc efflux transporter family members

Abstract

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 ZNT proteins based on their subcellular localization overlapping with TMEM163 and previous reports that certain 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 HEK-293 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. Native co-IP using mouse tissues confirmed the interactions while 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 cells expressing TMEM163 homodimers extruded zinc slightly less efficiently than cells expressing TMEM163/ZNT heterodimers. Cell surface biotinylation revealed a subtle change in the plasma membrane localization of TMEM163 upon co-expression with certain ZNT proteins, which possibly explains why zinc efflux is marginally different for TMEM163 homodimers than TMEM163/ZNT heterodimers. Overall, our results show that the interaction between TMEM163 and distinct ZNT proteins is functionally relevant and that their heterodimerization may serve to influence their zinc efflux activity within specific tissues or cell types. Research HighlightsO_LITMEM163 protein heterodimerizes with mammalian ZNT1, ZNT2, ZNT3 and ZNT4 zinc efflux transporters. C_LIO_LITMEM163 and ZNT proteins partially co-localize in their respective plasma membrane or subcellular compartments, suggesting distinct cellular roles as homodimers and heterodimers. C_LIO_LIThe zinc efflux activity of TMEM163 or ZNT protein homodimers did not markedly differ from their TMEM163/ZNT heterodimer counterparts. C_LIO_LIFunctional TMEM163/ZNT heterodimers give further credence to the role of TMEM163 as a bona fide member of the SLC30 protein family C_LI