Promotion of vesicular zinc efflux by ZIP13 and its implications for spondylocheiro dysplastic Ehlers-Danlos syndrome - PubMed (original) (raw)

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

Jeeyon Jeong et al. Proc Natl Acad Sci U S A. 2012.

Abstract

Zinc is essential but potentially toxic, so intracellular zinc levels are tightly controlled. A key strategy used by many organisms to buffer cytosolic zinc is to store it within vesicles and organelles.It is yet unknown whether vesicular or organellar sites perform this function in mammals. Human ZIP13, a member of the Zrt/Irt-like protein (ZIP) metal transporter family, might provide an answer to this question. Mutations in the ZIP13 gene, SLC39A13, previously were found to cause the spondylocheiro dysplastic form of Ehlers–Danlos syndrome (SCD-EDS), a heritable connective tissue disorder.Those previous studies suggested that ZIP13 transports excess zinc out of the early secretory pathway and that zinc overload in the endoplasmic reticulum (ER) occurs in SCD-EDS patients. In contrast,this study indicates that ZIP13’s role is to release labile zinc from vesicular stores for use in the ER and other compartments. We propose that SCD-EDS is the result of vesicular zinc trapping and ER zinc deficiency rather than overload.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

ZIP13 is a zinc transporter, and SLC39A13 expression is regulated by zinc. (A) Concentration-dependent zinc uptake (pmol⋅min−1⋅mg protein−1) in HEK293 cells overexpressing ZIP13 (▲) or vector-only control cells (●). n = 3; *P < 0.05. (B) Competition assays with HEK293 cells overexpressing ZIP13. 65Zn accumulation (pmol/mg protein) was measured in the presence of 10- or 50-fold molar excess of the metals indicated. C, control with no metals added. All metals were divalent cations except for Ag(I). Each data point represents mean values from three independent experiments. The effect of added zinc was much greater than that of other metals (P < 0.001). Other metals found to have some effect in these assays (P < 0.05) were Cu, Cd, Co, Ag, Mg, Ni, and Fe. (C) SLC39A13 expression under various zinc concentrations, with or without TPEN (12 h). Mean relative transcript levels detected by qRT-PCR are shown. n = 4; *P < 0.05. (D) Quantitation of ZIP13 protein levels under different zinc conditions. Untransfected HeLa cells were grown in basal medium or were treated for 12 h with 5 µM TPEN (low zinc) or 100 µM ZnCl2 (high zinc). ZIP13 protein levels were detected by immunoblots and normalized with total protein. Mean values are shown. n = 4; *P < 0.05. Error bars indicate SE.

Fig. 2.

Fig. 2.

Cells with loss of ZIP13 function. (A) Immunoblot with primary fibroblasts. Fibroblasts from an SCD-EDS patient (P3/II) and fibroblasts from a heterozygous parent (M2/II) were grown in basal medium, and total membranes were prepared for ZIP13 immunoblotting. (B) Immunofluorescence staining of ZIP13 in primary fibroblasts. M2/II and P3/II cells were grown under basal conditions and probed with anti-ZIP13 antibody. (C) FluoZin-3–stained images of primary fibroblasts showing labile zinc distribution. The cells were treated with 100 µM ZnCl2 for 12 h before staining. (D) Expression of SLC39A13 in HeLa cells treated with siRNA. HeLa cells were transiently transfected with nontargeting control siRNA (NT) or a pool of siRNAs targeting ZIP13 (siZIP13). Total RNA was isolated from the transfectants, and SLC39A13 levels were detected by qRT-PCR. Mean values are shown. n = 3; *P < 0.001. Error bars indicate SE. (E) Immunofluorescence staining of ZIP13 in NT and siZIP13 HeLa cells. (F) FluoZin-3 staining of NT and siZIP13 cells treated as in C.

Fig. 3.

Fig. 3.

ZIP13 does not localize to the ER or Golgi. Immunofluorescence colocalization with ZIP13 (red) and ER (CD3Δ-GFP, KDELR) or Golgi (Golgin97) markers (green) in HeLa cells.

Fig. 4.

Fig. 4.

Cytosolic zinc levels are decreased in ZIP13 loss-of-function cells. (A and B) Expression of MT1a (A) and MT2a (B) detected by qRT-PCR in primary fibroblasts. (C) Zap1-based reporter assays in HeLa cells cotransfected with GAL1UAS-luciferase (firefly), Renilla luciferase, nontargeting (NT) or ZIP13-targeting (siZIP13) siRNAs, and pGal4DBD-Zap1 or pGal4DBD alone and treated with different zinc conditions as indicated. Zap1-induced luciferase activity was normalized to Renilla luciferase and total protein. Mean values are shown. n = 3; *P < 0.05. Error bars indicate SE. (D) FRET ratio of siRNA-treated HeLa cells expressing cyto-ZapCY2 under basal conditions.

Fig. 5.

Fig. 5.

Zinc status of the early secretory pathway. (A and B) Endogenous ALP activities in fibroblasts from a heterozygous patient (M2/II) and from an SCD-EDS patient (P3/II). Cells were grown in basal conditions or treated with TPEN and/or supplemented zinc with or without pyrithione for 12 h before the assay. (C) ALP activities in siRNA-treated HeLa cells overexpressing TNAP. Nontargeting (NT) or ZIP13-targeting (siZIP13) siRNA transfectants were grown in basal conditions or treated with combinations of TPEN and/or supplementary ZnCl2 for 12 h before the assay. Mean values are shown. n = 4; *P < 0.05. Error bars indicate SE.

Fig. 6.

Fig. 6.

Induction of ATF6 activity in ZIP13 knockdown cells. HeLa cells were cotransfected with nontargeting (NT) or ZIP13-targeting (siZIP13) siRNA, 5xATF6-luciferase (firefly), and Renilla luciferase with or without ZnT7. Transfectants were treated with high zinc (100 µM ZnCl2) and/or tunicamycin (Tm) 12 h before the assays. 5xATF6-luciferase activity was normalized to Renilla luciferase and total protein. Inset shows luciferase activity detected under basal and high-zinc conditions. Mean values are shown. n = 3; *P < 0.05. Error bars indicate SE.

Fig. 7.

Fig. 7.

Proposed model of ZIP13 function. ZIP13 localizes to vesicular sites that buffer cytosolic zinc levels to provide zinc for use in other compartments. Uptake into the ZIP13 compartment competes with uptake into other compartments and with utilization in the cytosol and nucleus. Cells with reduced ZIP13 function accumulate zinc in vesicles, causing zinc deficiency in the cytosol and other organelles, such as the ER, and leading to ER dysfunction and stress. Blue circles represent zinc ions, and the white question marks denote unknown zinc transporters. ZnT5, ZnT6, and ZnT7 likely contribute to zinc uptake into the ER, although they are primarily Golgi transporters. ZIP7, which is localized to the ER, transports zinc out of the ER. The relationship of the ZIP13 compartment (black question mark) to known organelles remains to be determined.

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