Cell surface glycoproteins of CHO cells. I. Internalization and rapid recycling

The major cell surface proteins of Chinese hamster ovary (CHO) cells have been investigated after reacting cells at 40C with the membrane-impermeant reagent, trinitrobenzenesulfonate (TNBS). Immunoprecipitation and subsequent two-dimensional, sodiumdodecyl sulfate, polyacrylamide gel electrophoresis (SDS-PAGE) of proteins from derivatized cells that had been labelled previously with [3H]D-glucosamine or [3H]L-leucine showed that TNBS reacted with most of the high molecular weight (HMW) acidic glycoproteins that became labelled with iodine by the lactoperoxidase technique and that bind the lectin, wheat germ agglutinin (WGA). After warming the cells to allow endocytosis to proceed, molecule haptenized with trinitrophenol (TNP) groups were followed radio-chemically by means of [125I]anti-DNP antibodies. Within 15 min at 370C, a steady-state between surface and cytoplasmic label was reached, with about 65% of the hapten located internally. Recycling of internalized TNP groups back to the cell surface also occurred rapidly (t/sub 1/2/ ∼ 5 min). Our results are consistent with the view that the majority of plasma membrane glycoproteins are continuously being internalized and recycled at a high rate