A new method for detecting endocytosed proteins.
A new reagent, DPSgt, is described which has been designed to label cell surface proteins at 0 degree C. The reagent is easily made; it is water soluble and contains a reactive impermeant ester at one end, a tyrosine which can be radioiodinated at the other, and a disulphide in-between. The label can be removed from cells by cleaving the disulphide linkage in it with glutathione at 0 degree C. When cells are warmed to 37 degrees C between labelling and reduction, labelled proteins which are endocytosed acquire resistance to reduction. This provides a simple way of measuring the endocytosis of surface proteins. The intracellular pools of transferrin and LDL receptors in K562 cells and fibroblasts have been estimated. The results indicate that intracellular receptors are in non-reducing compartments, and that uptake of average cell surface (by non-coated pit processes) in K562 cells is small.
Bretscher
M S
MS
Medical Research Council, Laboratory of Molecular Biology, Cambridge, UK.
Lutter
R
R
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004151:Dipeptides; D004705:Endocytosis; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents; D007424:Intracellular Fluid / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D011973:Receptors, LDL / Q000378:metabolism; D011990:Receptors, Transferrin / Q000378:metabolism; D013388:Succinimides; D014407:Tumor Cells, Cultured / Q000378:metabolism
C059298:3,3'-dithiopropionyl 1-sulfosuccinimidyl 1'-glycyl-tyrosine; D004151:Dipeptides; D007202:Indicators and Reagents; D008565:Membrane Proteins; D011973:Receptors, LDL; D011990:Receptors, Transferrin; D013388:Succinimides