Abstract
The factors necessary to dissociate iron from transferrin in endocytic vesicles and to mobilize the iron across the vesicle membrane were studied in a preparation of endocytic vesicles markedly enriched in transferrin-transferrin receptor complexes isolated from rabbit reticulocytes. Vesicles were prepared with essentially fully saturated transferrin by incubating the reticulocytes with the protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone prior to incubation with 59Fe, 125I-transferrin with or without fluorescein isothiocyanate labeling. Initiation of acidification by the addition of ATP was sufficient to achieve dissociation of 59Fe from transferrin with a rate constant of 0.054 +/- 0.06 s-1. Mobilization of 59Fe out of the vesicles required, besides ATP, the addition of a reductant with 1 mM ascorbate, allowing approximately 60% mobilization at 10 min with a rate constant of 0.0038 +/- 0.0006 s-1. An NADH:ferricyanide reductase activity could be demonstrated in the vesicles with an activity of 7.1 x 10(-9) mol of NADH reduced per min/mg of vesicle protein. Both dissociation and mobilization were inhibited by N-ethylmaleimide, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, and monensin. Mobilization, but not dissociation, was inhibited by the permeant Fe(II) chelator alpha,alpha'-dipyridyl. The Fe(III) chelators deferoxamine, diethylenetriaminepentaacetic acid, and apotransferrin did not promote mobilization of dissociated iron in the absence of a reductant. This study establishes the basis for the cellular incorporation of iron through the endocytic pathway in which the endocytic vesicle membrane utilizes, in a sequential way, an acidification system, an iron reduction system, and an Fe(II) transporter system.
Highlights
From the Departamento de Biologia, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile and $Department of Medicine, Louisiana State University Medical School, Shreveport, Louisiana 71130
Reductase in Endocytic Vesicles-Initial exueriments indicated that the vesicles had NADH: ferricyanide reductase activity when assayed following the reduction of ferricyanide under the conditions described by Sun et al [17]
Acidification of Endocytic Vesicles-The acidification of the vesicles after the addition of ATP was assayed by the fluorescence quenching of FITC-labeled transferrin contained in the vesicles
Summary
From the Departamento de Biologia, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile and $Department of Medicine, Louisiana State University Medical School, Shreveport, Louisiana 71130. An NADH:ferricyanide reductase activity could be demonstrated in the vesicles with an activity of 7.1 x lo-’ mol of NADH reduced per min/mg of vesicle protein Both dissociation and mobilization were inhibited by N-ethylmaleimide, carbonyl cyanide. This study was undertaken to test the hypothesis that both ATP-mediated proton fluxes and iron reduction are required for iron dissociation from transferrin and translocation into the cytosol. 2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid; NEM, N-ethylmaleimide; BPS, bathophenanthroline disulfonate; DTPA, diethylenetriaminepentaacetic acid This study of iron transport in isolated endocytic vesicles establishes a model for iron entrance into the cytoplasm in which vesicle acidification, iron-transferrin dissociation, iron reduction, and iron translocation occur in a sequential manner
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.