Transferrin recycling endosome

After 1 hour of treatment, fluorescent transferrin can be localized in early endosomes and the endosomal-recycling compartment, whereas the cholera toxin B-subunit distinctly traffics to the trans-golgi network of living cells. In cells treated with holo-cholera toxin comprising the B-subunit and the catalytic A-subunit, the toxin would traffic further to the endoplasmic reticulum, which would enable the release of the toxic A-subunit into the cytoplasm. 

Zastrow MV, Kobilka BK (1992) Ligand-regulated internalization and recycling of human j02-adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors. J Biol Chem 267 3530-3538... 

Ligand (LDL, transferrin, etc.) Recycling receptor Retrograde vesicle Early endosome... 

D.P. Cemeus and A. Van der Ende. Apical and basolateral transferrin receptors in polarized BEWO cells recycle through separate endosomes. J Cell Biol. 114 1149-1158 (1991). 

Ren M, Xu G, Jinanbo Z, de Lemos-Chiarandini C, Adesnik M, Sabatini DD. Hydrolysis of GTP on rab 11 is required for the direct delivery of transferrin from the pericentriolar recycling compartment to the cell surface but not from sorting endosomes. Proc Natl Acad Sci USA 1998 95 6187-6192. 

Zobiack, N., Rescher, U., Ludwig, C., Zeuschner, D., and V. Gerke, 2003, The annexin 2/S100A10 complex controls the distribution of transferrin receptor-containing recycling endosomes. Mol Biol Cell. 14(12) 4896-908. 

It should be pointed out, however, that not all hormones dissociate from their receptor in the pH 5.5 environment of the endosome [24], Some hormone-receptor complexes require much lower pH values for dissociation to occur. Although not a peptide hormone, the iron-transport protein transferrin is a peculiar example of this phenomenon and should be pointed out. In this case, at the neutral pH of the extracellular fluid transferrin containing bound iron binds to its cell surface receptor and is internalized. In the low pH environment of the endosome, iron becomes dissociated from transferrin, but transferrin remains bound to its receptor. The transferrin receptor, with bound transferrin, is then recycled to the cell surface. With iron no longer bound to the transferrin, the transferrin readily dissociates from its receptor at the neutral pH of the extracellular fluid [25,26]. This mechanism provides for an efficient continual uptake of iron into cells. Unlike transferrin, however, in those instances where peptide hormones have been documented not to be dissociated from their receptor in the endosome compartment, the hormone and receptor are delivered to the lysosomes via fusion of the endosomes with lyso-somes, where both hormone and receptor are degraded [24,27]. The continuous degradation of the receptor with each round of RME eventually leads to a decrease in the number of receptors on the cell surface, a phenomenon called down-regulation. 

In the bloodstream, ferric iron binds tightly to circulating plasma transferrin (TF) to form diferric transferrin (FeTF). Absorption of iron into erythrocytes depends on basolateral membrane receptor-mediated endocytosis of FeTF by transferrin receptor 1 (TfR 1). FeTF binds to TfR 1 on the surface of erythroid precursors. These complexes invaginate in pits on the cell surface to form endosomes. Proton pumps within the endosomes lower pH to promote the release of iron into the cytoplasm from transferrin. Once the cycle is completed,TF and TfR 1 are recycled back to the cell surface. TF and TfR 1 play similar roles in iron absorption at the basolateral membrane of crypt enterocytes (Parkilla et al., 2001 Pietrangelo, 2002). 

Sipe, D. M. Jesurum, A. Murphy, R. F. Absence of sodium-potassium ATPase regulation of endosomal acidification in K562 erythroleukemia cells. Analysis via inhibition of transferrin recycling by low temperatures. J. Bio. Chem. 1991, 266, 3469-3474. 

Fig. 1. Principle of the in vitro fusion assay. After labeling the endocytic pathway with acridinium-transferrin (Ac-Tfn) labeled endosomes (donor) are enriched by density centrifugation. Recycling endosomes (acceptor) are purilied from unlabeled cells first by density centrifugation followed by immunoisolation using anti-Rabll coated magnetic beads. Transport of Ac-Tfn from the donor endosomes to acceptor recycling endosomes is performed at 3T and in the presence of cytosol and ATP. After washing the beads-bound membranes, transferred Ac-Tfn is measured by detecting acridinium cleavage in a luminometer.

The combined methods of preparing an endosome-enriched fraction labeled with acridinium-transferrin and specific immunoadsorption of unlabeled Rabll-positive recycling endosomes provide the basis of our in vitro fusion assay. Using this assay, transport of the physiological marker transferrin from labeled donor endosomes to immunoisolated acceptor recycling endosomes can be followed by measuring arrival of acridinium-transferrin to beads-bound endosomes as detected by lashlight-luminescence in a luminometer. 

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