Zinc transporter 2 (SLC30A2) can suppress the vesicular zinc defect of adaptor protein 3-depleted fibroblasts by promoting zinc accumulation in lysosomes

Abstract

Zinc accumulation in the lumen of cytoplasmic vesicles is one of the mechanisms by which cells can store significant amounts of this essential but potentially toxic biometal. Previous studies had demonstrated reduced vesicular zinc levels in fibroblasts from mutant mice deficient in adaptor protein 3 (AP-3), a complex involved in protein trafficking to late endosomes and lysosomes. We have observed a similar phenotype in the human fibroblastoid cell line, M1, upon small interference RNA-mediated AP-3 knockdown. A survey of the expression and localization of zinc transporter (ZnT) family members identified ZnT2, ZnT3 and ZnT4 as likely mediators of vesicular zinc accumulation in M1 cells. Expression of green fluorescence protein (GFP)-tagged ZnT2 and ZnT3 promoted accumulation of vesicular zinc as visualized using the indicator zinquin. Moreover, GFPZnT2 overexpression elicited a significant accumulation of zinc within mature lysosomes, which in untransfected M1 cells contained little or no chelatable zinc, and restored the zinc storage capability of AP-3-deficient cells. These results suggest that ZnT2 can facilitate vesicular zinc accumulation independently of AP-3 function, and validate the M1 fibroblastoid line as a human cell culture system amenable to the study of vesicular zinc regulation using techniques compatible with functional genomic approaches.

Promotion of vesicular zinc efflux by ZIP13 and its implications for spondylocheiro dysplastic Ehlers–Danlos syndrome

“…These vesicles have been referred to as "zincosomes" because their relationship to known compartments was unclear (26,51). Zincosomes were reported to be late endosomes (27,52), but this result later was shown to be an experimental artifact (53). On the other hand, the ZIP13 vesicles may result from endocytotic events, given that many ZIP transporters reside on the plasma membrane (15).…”

“…Nonetheless, the organellar identity of zincosomes is still unknown. Suggested roles for zincosomes in cellular zinc homeostasis include detoxifying excess zinc (27,53), buffering cytosolic zinc from transient perturbations of zinc homeostasis (26,49,51,54,55), and storing readily accessible zinc for later use under zinc deficiency (54,56). The zinc transporters that move zinc into and out of zincosomes also are unknown.…”

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

“…Thus, in both untransfected and transfectant cells, no toxicity could be attributed to extracellular ZnSO 4 concentrations of 0.1-100 M. Based on these findings, an extracellular zinc concentration of 75 M was further used upon flow cytometry and fluorescence microscopy experiments in order to determine vesicular zinc accumulation in WT and mutant ZnT2 transfectants. This very same concentration of ZnSO 4 that was previously used in vesicular Zinquin accumulation experiments (7,13,37) (Fig. 8A, iv-vi) or co-transfected with both WT ZnT2-YC and WT ZnT2-YN (Fig.…”

“…Cells showing fluorescence that was above the autofluorescence level were considered Zinquin-positive cells or PI-positive cells. Based on our zinc titration results and previously published vesicular Zinquin accumulation experiments (7,13,37), an extracellular concentration of 75 M ZnSO 4 was used in order to evaluate vesicular zinc accumulation in WT and mutant ZnT2 MCF-7-transfected cells using flow cytometry and Zinquin fluorescence microscopy.…”

“…ZnT2, ZnT3, ZnT4, and ZnT8 are expressed in a cell-and tissue-specific manner; ZnT2 and ZnT4 are highly expressed in the mammary gland and small intestine. ZnT2 is predominantly localized in cytoplasmic vesicles, such as late endosomes (7,8), and secretory vesicles in specialized secretory epithelia, such as the mammary gland, prostate, and pancreas (9). ZnT5, ZnT7, and ZnT6 (10) are widely expressed and localized in the early secretory pathway, including the endoplasmic reticulum (ER), 3 Golgi, and cytoplasmic vesicles (11)(12)(13).…”