FIG. 4.

The processed ZIP4 carboxyl-terminal domain preferentially accumulates in the apical membrane in MDCK and CaCo2 cells, and these cells show a dramatically decreased dose response for zinc induction of Mt1 expression. (A) Pools of stably transfected MDCK and CaCo2 cells expressing FLAG-ZIP4-HA were grown in transwell plates until polarized and then cultured in medium containing either 10% normal FBS (N) or 10% Chelex-treated FBS (CX) for 48 h before cell surface biotinylation assays were performed. The biotinylation reagent (sulfo-NHS-SS-biotin) was added to either the apical (Ap) or basolateral (Bl) compartment of the transwell plate, as described in Materials and Methods. Solubilized proteins were captured using streptavidin beads and analyzed by Western blotting using an anti-HA antibody (αHA). Total lysates refers to aliquots of the input biotinylated proteins from the apical surface before avidin capture. The processed carboxyl-terminal peptide of ZIP4 was preferentially biotinylated in the apical membrane. ZnT5 and NaK-ATPase α1 antisera were used as controls for input and for efficiency of basolateral membrane biotinylation, respectively. (B) Nontransfected control MDCK cells (−) and pools of MDCK cells stably transfected with the FLAG-ZIP4-HA expression vector were cultured for 48 h in medium containing either 10% normal FBS (N) or 10% Chelex-treated FBS (CX). Cells were then treated from the apical chamber with the indicated concentrations of ZnSO₄ (0 to 20 μM) for 6 h. Total RNA (20 μg) was isolated and fractionated by formaldehyde-agarose gel electrophoresis, transferred to nylon membranes, and hybridized with mouse Mt1 and Slc39a4 cRNA probes. Hybrids were detected by autoradiography. Gels were stained with acridine orange as a control for RNA integrity and loading (bottom panel). The relative abundance of Mt1 mRNA without zinc was defined as 1.0-fold induction, and the change in induction relative to that sample is shown below each lane (top panel).