We have demonstrated a requirement for cellular ATP in the receptor-mediated endocytosis of transferrin. This has been accomplished using a novel assay for endocytosis based on acquisition of resistance to the membrane impermeable reducing agent, glutathione (GSH). Diferric-transferrin was conjugated to biotin via a cleavable disulfide bond and iodinated. Internalization of 125I-biotin-S-S-transferrin (125I-BSST) was quantitated by adsorption to avidin-Sepharose after treatment of cells with GSH. Receptor-mediated endocytosis of 125I-BSST was severely inhibited in ATP-depleted cells. Similar results were obtained when ATP was depleted by incubation of cells either under a N2-atmosphere or in the presence of NaN3 and NaF. The latter treatment, alone, also resulted in a loss of surface transferrin receptors which could not be correlated to reductions in cellular ATP. In contrast to the acquisition of GSH resistance, the apparent internalization of 125I-BSST as assessed by inaccessibility to antitransferrin antibodies reached control levels in ATP-depleted cells. Our biochemical and morphological data suggested that, although ATP is required for receptor-mediated endocytosis, in ATP-depleted cells ligands can become efficiently sequestered into deeply invaginated pits that are inaccessible to large probes such as antibodies, but remain accessible to small molecules such as GSH.
Article|
December 01 1990
ATP is required for receptor-mediated endocytosis in intact cells.
S L Schmid,
S L Schmid
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
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L L Carter
L L Carter
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
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S L Schmid
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
L L Carter
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
Online ISSN: 1540-8140
Print ISSN: 0021-9525
J Cell Biol (1990) 111 (6): 2307–2318.
Citation
S L Schmid, L L Carter; ATP is required for receptor-mediated endocytosis in intact cells.. J Cell Biol 1 December 1990; 111 (6): 2307–2318. doi: https://doi.org/10.1083/jcb.111.6.2307
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