The human (h) zinc transporter, ZIP4, is the primary zinc importer in the intestine. hZIP4 is also expressed in various organs such as the pancreas and brain. Dysfunction of hZIP4 can result in the zinc deficiency disease acrodermatitis enteropathica (AE). AE can disrupt the digestive and immune system homeostasis. Structure-function studies of hZIP4 have significantly been hindered by the absence of a robust heterologous expression system. Here, we report the heterologous expression of hZIP4 in Saccharomyces cerevisiae. A wild type and a mutant S. cerevisiae strain, in which the endogenous zinc transporters are deleted, were used to test the expression and localization of an hZIP4-GFP fusion protein. A full-length hZIP4-GFP and a truncated membrane domain only (mhZIP4-GFP) protein were successfully expressed and targeted to the plasma membrane in yeast. hZIP4 encodes a large N-terminal extracellular domain (ECD). Mutations in the ECD can lead to AE. Previous structural studies showed that the hZIP4-ECD has two structurally independent subdomains and a dimer centered at the Proline-Alanine-Leucine (PAL) motif. These results led to the hypothesis that ZIP4-ECD contributes to the structural organization of the protein. To explore the role of the ECD in hZIP4 dimerization, fluorescence correlation spectroscopy (FCS) was used to quantify the oligomeric state of hZIP4. The results show that the transmembrane domains are sufficient to dimerize hZIP4.

Creator

• Liu, Yuting

Contributeurs

• Dempski, Robert E.
• Young, Eric M.
• Argüello, José M.
• Burdette, Shawn C.

Degree

• PhD

Unit

• Chemistry & Biochemistry

Publisher

• Worcester Polytechnic Institute

Identifier

• etd-70721

Advisor

• Dempski, Robert E.

Committee

• Burdette, Shawn C.
• Argüello, José M.
• Young, Eric M.

Defense date

• 2022-05-17

Year

• 2022

Date created

• 2022-07-11

Resource type

• Dissertation

Source

• etd-70721

Rights statement

• In Copyright