Abstract
The iron-responsive element binding protein (IRE-BP) interacts with specific sequence/structure motifs (iron-responsive elements) within the mRNAs encoding ferritin and the transferrin receptor and thereby post-transcriptionally regulates the expression of these two proteins involved in cellular iron homeostasis. The activity of the IRE-BP is itself regulated by iron such that when cells are treated with an iron source, the RNA binding activity is decreased. The expression of recombinant human IRE-BP in murine cells has been examined as have the expressions of the endogenous IRE-BP of both human and rabbit cells. In all cases, iron down-modulated the RNA binding activity of the IRE-BP, but in no instance was this decrease in activity accompanied by a decrease in the level of the protein as judged by quantitative Western blots. Moreover, the rate of synthesis of the IRE-BP and its rate of degradation have been found to be unaltered by iron manipulation of cells in culture. Consistent with IRE-BP regulation occurring post-translationally, the iron regulation of its activity was found to be unaffected by cycloheximide. These data are discussed in terms of a model of IRE-BP regulation involving the modification of the protein's iron-sulfur center.
Highlights
The iron-responsive element binding protein (IREBP) interacts with specific sequence/structure motifs within the mRNAs encoding ferritin and the transferrin receptor and thereby post-transcriptionally regulates the expression of these two proteins involved in cellular iron homeostasis
While these studies provide a likely mechanism for the post-translational regulation of this protein, it remains to be directly demonstrated that iron manipulations do not result in changes in thetotal levels of the ironresponsive elements (IREs)-BPthrough alteration of either its rateof synthesis ordegradation
Lysates of cells grown in the presence of an iron source such as hemin orin the presence of the iron chelator, desferrioxamine,can beusedin gel retardationstudies witha radiolabeled IRE-containing RNA
Summary
Vol 267, No.[34], Issue of December 5,pp. 24466-24470,199’2 Printed in U.S.A. Iron Regulates the Activity of the Iron-responsive Element Binding Protein without ChangingIts Rate of Synthesis or Degradation*. Consistent with IRE-BP regulation occurring post-translationally, the iron regulation of its activity was found to be unaffected by signal that istransduced into regulated binding to IREs (1113) While these studies provide a likely mechanism for the post-translational regulation of this protein, it remains to be directly demonstrated that iron manipulations do not result in changes in thetotal levels of the IRE-BPthrough alteration of either its rateof synthesis ordegradation. We report the use of both monoclonal antibodies to epitope-tagged recombinant IRE-BP andpolyclonal cycloheximide These data are discussed in terms of a sera that recognize native protein, and we demonstrate that model of IRE-BP regulation involving the modification levels of IRE-BP arestable after treatments of cells with the of the protein’s iron-sulfur center. The abbreviations used are: TfR,transferrin receptor; IRE, ironresponsive element; IRE-BPI iron-responsive element binding protein; 2-ME, 2-mercaptoethanol
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