Abstract
The ZIP superfamily of transporters plays important roles in metal ion uptake in diverse organisms. There are 12 ZIP-encoding genes in humans, and we hypothesize that many of these proteins are zinc transporters. In this study, we addressed the role of one human ZIP gene, hZIP1, in zinc transport. First, we examined (65)Zn uptake activity in K562 erythroleukemia cells overexpressing hZIP1. These cells accumulated more zinc than control cells because of increased zinc influx. Moreover, consistent with its role in zinc uptake, hZIP1 protein was localized to the plasma membrane. Our results also demonstrated that hZIP1 is responsible for the endogenous zinc uptake activity in K562 cells. hZIP1 is expressed in untransfected K562 cells, and the increase in mRNA levels found in hZIP1-overexpressing cells correlated with the increased zinc uptake activity. Furthermore, hZIP1-dependent (65)Zn uptake was biochemically indistinguishable from the endogenous activity. Finally, inhibition of endogenous hZIP1 expression with antisense oligonucleotides caused a marked decrease in endogenous (65)Zn uptake activity. The observation that hZIP1 is the major zinc transporter in K562 cells, coupled with its expression in many normal cell types, indicates that hZIP1 plays an important role in zinc uptake in human tissues.
Highlights
Zinc is an essential nutrient to all organisms because it is a required catalytic and/or structural cofactor for hundreds of zinc-dependent enzymes and other proteins such as transcription factors
Our results demonstrated that hZIP1 is responsible for the endogenous zinc uptake activity in K562 cells. hZIP1 is expressed in untransfected K562 cells, and the increase in mRNA levels found in hZIP1-overexpressing cells correlated with the increased zinc uptake activity
Given the ubiquitous expression of hZIP1 in human tissues, we propose that hZIP1 is the major zinc transporter for many human cell types
Summary
DNA Manipulations—An hZIP1 cDNA clone was isolated from a prostate library using high throughput PCR screening (Genome Systems Inc.) and sequenced in its entirety. HZIP1 was epitope-tagged at its amino terminus with one copy of the hemagglutinin antigen (HA) by PCR to generate CMV-HA-hZIP1 This fragment was digested with HindIII and XbaI, cloned into the pRc-CMV vector, and confirmed by sequencing. The cells were resuspended in uptake buffer and incubated for 10 min in a shaking 37 °C water bath. The cells were mixed with an equal volume of prewarmed uptake buffer containing the specified concentration of 65ZnCl2 (Amersham Pharmacia Biotech) and incubated for 15 min unless indicated otherwise. GFP/fluorescein-positive cells were grown for an additional 24 h before mRNA was isolated or 65Zn uptake assays were performed. HZIP1 and glyceraldehyde-3phosphate dehydrogenase fragments were amplified from the cDNA templates using 35 cycles These conditions were found to be in the quantitative range for detection of products based on input template concentration. The products were analyzed by agarose gel electrophoresis, stained with ethidium bromide, and photographed under UV light with the CCD camera. -Actin primers were used to confirm similar levels of input cDNA in each PCR sample
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