In Situ Dimerization of Multiple Wild Type and Mutant Zinc Transporters in Live Cells Using Bimolecular Fluorescence Complementation

Abstract

Background: Zinc transporters (ZnTs) presumably form dimers, thereby modulating zinc transport activity. Results: Bimolecular fluorescence complementation (BiFC) revealed ZnT1-4, ZnT7 homodimers and ZnT5-ZnT6 heterodimers. Conclusion: BiFC pinpointed WT and mutant ZnT2 dimerization in live cells. Significance: BiFC provides the first in situ evidence for the subcellular localization of WT and mutant ZnT2 dimers in live cells, thereby establishing the molecular basis underlying zinc deficiency.

Essential Role for Zinc Transporter 2 (ZnT2)-mediated Zinc Transport in Mammary Gland Development and Function during Lactation

“…Lactating ZnT2-null Mice Accumulate Zinc and Have Profound Defects in Morphology and Impaired MEC Differentiation-Consistent with results in nulliparous ZnT2ko mice and previous reports in breastfeeding women with heterozygous mutations in ZnT2 (11,17), overall zinc status as measured by plasma (wt, 13.08 mol/liter Ϯ 1.4; ZnT2ko, 13.95 mol/liter Ϯ 1.3) and liver (wt, 32.38 g Zn/g of tissue Ϯ 2.6; ZnT2ko, 31.78 g Zn/g of tissue Ϯ 4.5) zinc concentration was not altered in the lactating ZnT2ko mice. Additionally, similar to previous reports in rats and mice (31,32), ZnT2 was localized intracellularly and associated with the apical membrane of MECs in the mammary gland during lactation (Fig.…”

“…ZnT2 is expressed in the mammary gland (47), and previous studies have focused on its role in importing zinc into vesicles (10, 13) for secretion into milk during lactation (11,16,17). ZnT2 is also found in mitochondria and vesicles in non-lactating mammary glands; however, the physiological role of ZnT2-mediated zinc transport beyond that of zinc secretion into milk is almost entirely unknown.…”

“…9A). Because women with mutations in ZnT2 have a much greater (50 -90%) reduction in milk zinc concentration (10,11,13,16,17), we expected to see a greater loss of zinc transfer into milk in ZnT2ko mice. Given this unexpected finding, we tested the hypothesis that ZnT4 expression was augmented to compensate for the lack of ZnT2, as we and others (24,46) have shown that ZnT4 is important for zinc secretion into milk.…”

Heterodimerization, Altered Subcellular Localization, and Function of Multiple Zinc Transporters in Viable Cells Using Bimolecular Fluorescence Complementation

“…Once the two target proteins undergo a close physical interaction (less than 15 nm), this facilitates the non-fluorescent fragments of YFP to associate and refold, thereby leading to the resumption of YFP fluorescence (24). This sensitive bioassay enabled us to detect high fluorescence levels, which were indicative of homodimer formation of various ZnTs, including ZnT1-4 and ZnT7 (22). Furthermore, this BiFC assay allowed us to pinpoint the precise subcellular localization of ZnT5 and ZnT6 heterodimers in live cells (22).…”

“…Furthermore, using epitope tagging and co-immunoprecipitation, ZnT5 and ZnT6 were shown to form a stable heterodimer (21), although more sensitive tools were needed to corroborate ZnT dimerization and to follow the subcellular localization of these dimers in live cells. In this respect, we recently applied the bimolecular fluorescence complementation (BiFC) technique in order to provide direct visual evidence for the in situ dimerization of wild type (WT) and mutant ZnTs in live cells at their established intracellular organelles (22). The BiFC technique, which was originally devised to visualize specific protein-protein interactions in live cells (23), is based on the principle of tagging two proteins with two non-fluorescent halves of a fluorescent protein, such as yellow fluorescence protein (YFP).…”