TMEM163 (ZNT11) protein interacts with distinct ZNT efflux transporters

Abstract

Our recent work showed that TMEM163 is a zinc efflux transporter that belongs to the cation diffusion facilitator (CDF) family of proteins. We propose that the TMEM163 be now called ZNT11 as a new member of the Group 1 CDF family ZNT efflux proteins that include 10 mammalian zinc transporters, ZNT1‐ZNT10. Accordingly, we hypothesize that TMEM163/ZNT11 interacts with at least one of the ZNT proteins, namely ZNT1 and ZNT2, based on its structural and functional characteristics. To show that TMEM163/ZNT11 interacts with either ZNT1 or ZNT2, we cloned their cDNAs into mammalian expression constructs containing either Myc‐DDK or HA peptide tag. Using HEK‐293 cells, we individually transfected TMEM163/ZNT11, ZNT1, and ZNT2 constructs as negative controls, while we co‐transfected TMEM163/ZNT11 and ZNT1, or TMEM163/ZNT11 and ZNT2. As a positive control, we co‐transfected HA‐tagged TMEM163/ZNT11 with a Myc‐DDK‐tagged TMEM163/ZNT11 construct in HEK‐293 cells, based on a previous report that its rodent counterpart exists as a homodimer. We then used a co‐immunoprecipitation assay using either anti‐HA‐ or anti‐DDK‐bound agarose beads to pull down one of the target proteins. Western blot analysis showed that TMEM163/ZNT11 physically bound ZNT1 or ZNT2 protein. To determine the functional relevance of the interaction, we performed zinc flux assays using two zinc‐specific fluorescence dyes, Fluozin‐3 (high affinity, membrane impermeable) and Newport Green (low affinity, membrane permeable) following single‐ and co‐transfection of TMEM163/ZNT11, ZNT1, and ZNT2 constructs in HeLa cells. Our results confirmed that homodimers of TMEM163/ZNT11, ZNT1, and ZNT2 proteins transport zinc out of the cells, but that the efflux activity of homodimer TMEM163/ZNT11 proteins varied slightly in magnitude when compared with heterodimers of TMEM163/ZNT11 and ZNT1, or TMEM163/ZNT11 and ZNT2 proteins. These results suggest that the interaction between TMEM163/ZNT11 and distinct ZNT proteins is physiologically relevant and may serve to modify the transport activity of ZNT protein interactor. Overall, our investigations showed for the first time that TMEM163/ZNT11 forms functional heterodimers with ZNT1 and ZNT2 proteins. Thus, TMEM163/ZNT11 by itself, or in combination with one of these specific ZNT proteins, may play a crucial role in maintaining intracellular zinc homeostasis in specific cell types. Support or Funding Information This work is funded by NIH R15 NS101594.