Zinc transporter-2 (ZnT2) variants are localized to distinct subcellular compartments and functionally transport zinc

Abstract

ZnT2 (zinc transporter-2) expression is restricted to tissues with unique zinc requirements such as mammary and prostate glands. We previously determined that ZnT2 plays a major role in zinc export from mammary glands, as women with a mutation in the gene encoding ZnT2 (SLC30A2) had an approximately 75% reduction in milk zinc concentration. Two distinct human ZnT2 isoforms (approximately 42 and 35 kDa) are predicted to result from alternative splicing of SLC30A2. We examined the localization and function of each ZnT2 isoform, in cells generated to express ZnT2-HA (haemagglutinin) fusion proteins. The 42 kDa isoform was localized primarily to the endosomal/secretory compartment and overexpression resulted in increased zinc vesicularization. In contrast, the 35 kDa isoform is associated with the plasma membrane. Importantly, zinc transport was higher in cells over-expressing each isoform, indicating that both proteins are functional. Endogenous expression of the secretory vesicle-associated ZnT2 isoform predominates in mammary cells and expression is higher in secreting cells, whereas the smaller isoform plays a minor role in zinc export, directly reflecting the secretory function of the mammary gland. Together our data shed further light on the complex integration of cellular zinc transport mechanisms, which may be facilitated by multiple isoforms of specific zinc transporters with unique cellular functions.

Figure 1. The ZnT2 long isoform is localized to the secretory compartment in mammary epithelial cells

(A) Representative immunoblot of crude membrane proteins (100 μg protein/lane) from HC11 cells transfected with pcDNA3.1 (lane 1, mock-transfected control) or cells expressing the 42 kDa ZnT2 HA-fusion protein (lanes 2–3) detected with HA antibody. (B) Confocal micrographs of cells transfected with pcDNA3.1-longHA to express an HA-tagged 42 kDa ZnT2 fusion protein (ZnT2 long–HA) detected with HA antibody and visualized with Alexa Fluor^® 488-conjugated anti-mouse IgG. Confocal micrographs illustrate that the long ZnT2 isoform (green) resides in distinct vesicular compartments and co-localizes (merge, yellow) with M6PR (red, endosome marker) and VAMP8 (red, exocytotic vesicle marker). Asterisks represent individual transfected cells (n = 3 and 6 cells expressing ZnT2 long-HA fusion protein). Bar, 20 μm. (C) Accumulation of Zn into vesicles was assessed using FluoZin-3 fluorescence in cells transfected to over-express the long ZnT2 isoform (ZnT2 long–HA) and compared with mock-transfected cells. Data represent mean percentage fluorescence relative to mock-transfected cells ±S.D. (n = 6–8 samples/group). *P < 0.05, a significant effect of over-expressing ZnT2 isoform on Zn vesicularization.

Figure 2. ZnT2 localized to vesicles traffics to the plasma membrane and facilitates Zn secretion

(A) Confocal micrographs documented co-localization of the long ZnT2 isoform (ZnT2 long-HA; green) with pan-cadherin (red) illustrating localization (yellow) at the plasma membrane in secreting cells. Bar, 20 μm. (B) Expression of ZnT2 long-HA (ZnT2-HA) resulted in significantly higher Zn secretion from mammary cells relative to mock-transfected cells. There was no effect of the HA-tag (ZnT2-no HA) on Zn secretion observed. *P < 0.05, a significant effect of over-expressing the long ZnT2 isoform on Zn secretion.

Figure 3. The short ZnT2 isoform is localized to the plasma membrane in mammary epithelial cells

(A) Representative immunoblot (IB) of crude membrane proteins (100 μg protein/lane) from HC11 cells transfected with pcDNA3.1 (lane 1, mock-transfected control) or cells expressing the 35 kDa ZnT2 short–HA (lanes 2–3) detected with HA antibody. (B) Confocal micrographs of cells transfected with pcDNA3.1-shortHA, to express ZnT2 short–HA, detected with HA antibody and visualized with Alexa Fluor^® 488-conjugated anti-mouse IgG. Confocal micrographs illustrate detection of the ZnT2 short-HA at the cell periphery (arrows); COX IV (cytochrome c oxidase subunit IV; red) was utilized for spatial orientation. Asterisks represent individual transfected cells (n = 5 cells expressing ZnT2 short-HA). Bar, 10 μm. (C) Plasma membrane association was verified by immunoblot of cell surface proteins isolated from cells expressing the 35 kDa ZnT2 short-HA, captured with Ultralink Neutravidin, detected with HA antibody. Non-specific binding to avidinated beads was assessed in proteins captured from cells not exposed to biotin (—) and compared with biotinylated cell surface proteins.

Figure 4. Three specific ZnT2 endogenous isoforms are detected in mammary epithelial cells

(A) Representative immunoblots (IB) of crude membrane fractions (50 μg protein/lane) isolated by ultracentrifugation from non-secreting mammary epithelial cells. Crude membrane proteins were separated by electrophoresis and ZnT2 was detected by immunoblotting with pre-immune rabbit serum (lanes 1–2) or ZnT2 antibody (lanes 3–4). (B) To verify antibody specificity to each isoform, crude membrane proteins isolated from cells transfected with mismatched control siRNA (lanes 1–2) and cells transfected with SLC30A2-specific siRNA (lanes 3–4) were immunoblotted with ZnT2 antibody. Both methods illustrate specific detection of three ZnT2 proteins at ~55, ~42 and ~35 kDa. Equal loading was verified by immunoblotting for β-actin. (C) Densitometric analysis of immunoblot illustrating a significant effect of siRNA-mediated SLC30A2 attenuation on all three ZnT3 isoforms. * P < 0.05, a significant effect of SLC30A2 attenuation on isoform abundance.

Figure 5. Confocal microscopy identified specific sub-cellular compartments associated with endogenous ZnT2 isoforms in secreting mammary epithelial cells

HC11 cells were treated with prolactin and cortisol for 24 h to cause differentiation to a secreting phenotype. Double-immunofluorescence imaging of ZnT2 (green) and sub-cellular markers (red) in secreting mammary epithelial cells illustrating co-localization (merge, yellow) with M6PR (late endosome/secretory vesicle marker), VAMP8 (exocytotic vesicle marker) and to a lesser extent with PDI (endoplasmic reticulum marker). Minimal localization at the cell periphery was occasionally observed (arrows). No co-localization between ZnT2 isoforms and lysozomes (Lysotracker Red) or recycling endosomes (Transferrin) was observed in secreting mammary epithelial cells. Bars, 10 μm.

Figure 6. Cell surface biotinylation detects the expression of the endogenous 35 kDa ZnT2 isoform and a minimal amount of the 42 kDa ZnT2 isoform at the plasma membrane in secreting mammary epithelial cells

Representative immunoblot of plasma membrane-associated proteins captured with Ultralink Neutravidin detected with ZnT2 antibody. Non-specific binding to avidinated beads was assessed in proteins captured from cells not exposed to biotin (—; lanes 1–3) and compared with biotinylated cell surface proteins (+; lanes 4–6). Distinct localization of the 35 kDa isoform at the plasma membrane was routinely detected, whereas spurious and variable abundance of the 42 kDa isoform was observed.

Figure 7. Enhanced Zn accumulation and secretion is associated with higher abundance of the vesicular ZnT2 isoform in secreting mammary epithelial cells

(A) Representative immunoblot of ZnT2 associated with the crude membrane fraction isolated from mammary epithelial cells with a non-secreting and secretory phenotype (100 μg protein/lane; n = 3 samples/phenotype). Immunoblotting illustrated that a secretory phenotype was associated with greater abundance of the vesicular ZnT2 isoform, whereas abundance of the plasma membrane-associated ZnT2 isoform was negligible and not related to phenotype. Relative sample loading was visualized by immunoblotting for β-actin. (B) Zinc accumulation into vesicular pools was detected by confocal microscopy and quantified by fluorimetry in non-secreting and secreting mammary cells. Data represent mean fluorescence/μg of protein ± S.D. *P < 0.05, a significant effect of phenotype on Zn accumulation. (C) Zinc secretion over 120 min was measured in secreting and non-secreting mammary cells pre-loaded with [⁶⁵Zn] (n = 6 samples/phenotype per time). High-resolution confocal micrographs permit the visualization of increased vesicluarized Zn pools in secreting cells. *P < 0.05, a significant effect of phenotype on Zn secretion.