Quantifying the Oligomeric State of hZIP4 on the Surface of Cells

Abstract

The human (h) zinc transporter ZIP4 is expressed on the plasma membrane and functions to increase cytosolic zinc levels. Mutations in hZIP4 cause the disease acrodermatitis enteropathica. Dysfunction in the regulation of hZIP4 has also been indicated in solid tissue cancers, including pancreatic and prostate cancer. Although structural studies of the extracellular domain and computational modeling of the membrane domain suggest hZIP4 exists as a dimer, the oligomerization status of hZIP4 in the plasma membrane of mammalian cells has not been directly quantified in vivo. Here, the oligomeric state of hZIP4 expressed in HEK293 cells was quantified using fluorescence correlation spectroscopy. hZIP4 was tagged with eGFP, and by comparing brightness values (ε) of monomer and tandem eGFP constructs to that of an hZIP4/eGFP, we show that hZIP4 is a dimer. Determining that hZIP4 is a dimer is an important step toward understanding the function and processing of the protein, which can provide more insight into how diseases affected by hZIP4 occur and can be managed.

Figure 1.

hZIP4 is functional with the eGFP tag. HEK293 cells were transfected with either the empty vector (pcDNA3.1) as a negative control or hZIP4/eGFP (black or dashed bar, respectively). After 48 h, cells were lysed and incubated with 5 μM ⁶⁵Zn for 10 min. The cells were then washed, and the amount of ⁶⁵Zn that accumulated was measured. This is a representative data set in which n = 8; a total of three independent experiments were completed with similar results. Error bars here represent the standard error of the mean. Data were found to be significant using a t test with p < 0.05.

Figure 2.

Confocal microscopy showing plasma membrane localization of proteins. HEK293 cells were transfected with either (A and B) monomeric CD-86/eGFP, (C and D) tandem CD-86/eGFP/eGFP, or (E–H) hZIP4/eGFP. After 24 h, cells were imaged using confocal microscopy. Images A–F represent the focus on the center of a cell on the z-axis, while images G and H show the focus on the upper plasma membrane, where FCS measurements were taken. Images G and H also demonstrate the high level of expression of hZIP4/eGFP on the cell membrane. Images B, D, F, and H show an overlay of fluorescence excitation with a DIC image of the cells.

Figure 3.

Fluorescence correlation spectroscopy (FCS) autocorrelation curves of eGFP-tagged membrane proteins. FCS recordings were made on the plasma membrane of transiently transfected HEK293 cells expressing the indicated eGFP-tagged proteins. These graphs are representative examples taken from one of three independent studies with multiple samples. (A) Fluorescence intensity fluctuations from which recordings outside the range of 50–250 kHz were excluded. (B) Autocorrelation curve of FCS data (red line) with a model curve fitting a two-dimensional two-component model (blue line). Diffusion times for the fast (τ_D1) and slow (τ_D2) components are shown.