ATP is required for receptor-mediated endocytosis in intact cells.
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.
Schmid
S L
SL
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
Carter
L L
LL
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000693:Anaerobiosis; D001386:Azides / Q000494:pharmacology; D001710:Biotin; D002460:Cell Line; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism / Q000648:ultrastructure; D004705:Endocytosis / Q000187:drug effects; D005978:Glutathione / Q000494:pharmacology; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011990:Receptors, Transferrin / Q000187:drug effects / Q000378:metabolism*; D019810:Sodium Azide; D012969:Sodium Fluoride / Q000494:pharmacology; D014168:Transferrin / Q000378:metabolism*
D001386:Azides; D011990:Receptors, Transferrin; D014168:Transferrin; D001710:Biotin; D000255:Adenosine Triphosphate; D012969:Sodium Fluoride; D019810:Sodium Azide; D005978:Glutathione