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Biophysical and cell biological studies characterizing the vertebrate iron exporter ferroportin

Citation

Rice, Adrian Edward (2009) Biophysical and cell biological studies characterizing the vertebrate iron exporter ferroportin. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-05192009-163326

Abstract

Mammalian iron homeostasis is maintained by an intricate network of diverse proteins that constantly survey systemic iron levels and carefully regulate the uptake of iron from the diet. Control of this uptake is critically important because once iron is absorbed, mammals have no regulated mechanism for its removal. The portal through which iron enters the body is ferroportin, a multipass membrane protein expressed on the basolateral membrane of epithelial cells in the duodenum. The iron export function of ferroportin is primarily regulated by the serum peptide hormone hepcidin, which is secreted from the liver when systemic iron levels are high. Hepcidin acts as a negative regulator of iron uptake by binding to ferroportin at the cell surface and inducing its internalization and degradation. Genetic defects in ferroportin, hepcidin, or the proteins involved with sensing systemic iron levels lead to iron overload diseases known as hereditary hemochromatosis. Using the tools of biophysics and cell biology, we sought to study ferroportin and its interaction with hepcidin in order to better understand this critical bottleneck in iron uptake and how genetic defects within ferroportin might lead to disease. We developed the first protocols for the overexpression, detergent-solubilization, and purification of recombinant ferroportin. We determined that detergent-solubilized ferroportin is a monomer capable of binding hepcidin in vitro. We characterized the expression and subcellular localization of ferroportin in mammalian tissue culture and determined that both the amino- and carboxy-termini of ferroportin are cytosolic. We developed cell-based assays for the hepcidin-induced internalization of ferroportin and used these to characterize the route of internalization from the plasma membrane through early endosomes to degradative lysosomal compartments. Using live-cell imaging techniques, we showed that this internalization depended on intact microtubules. We expanded this cell-biological study to include sixteen disease-related ferroportin mutants and reported that each mutant was expressed on the plasma membrane like wild-type ferroportin, but that only a subset of the mutants were capable of being internalized by hepcidin. These studies form a foundation for future biophysical and cell-biological studies of ferroportin function.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:ferroportin; hemochromatosis; hepcidin; live-cell imaging; membrane protein
Degree Grantor:California Institute of Technology
Division:Biology
Major Option:Biochemistry and Molecular Biophysics
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Bjorkman, Pamela J. (co-advisor)
  • Rees, Douglas C. (co-advisor)
Thesis Committee:
  • Chan, David C. (chair)
  • Rees, Douglas C.
  • Jensen, Grant J.
  • Gray, Harry B.
  • Bjorkman, Pamela J.
Defense Date:29 April 2009
Record Number:CaltechETD:etd-05192009-163326
Persistent URL:http://resolver.caltech.edu/CaltechETD:etd-05192009-163326
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:1883
Collection:CaltechTHESIS
Deposited By: Imported from ETD-db
Deposited On:29 May 2009
Last Modified:26 Dec 2012 02:43

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