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

Abstract

The major cell surface proteins of Chinese hamster ovary (CHO) cells have been investigated after reacting cells at 4 °C with the membrane-impermeant reagent, trinitroben-zenesulfonate (TNBS). Immunoprecipitation and subsequent two-dimensional, sodium-dodecyl 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, molecules haptenized with trinitrophenol (TNP) groups were followed radiochemically by means of [ 125I]anti-DNP antibodies. The half-life for internalization of proteins tagged with either [ 125I]anti-DNP IgG or Fab averaged about 5 min. A similar result was obtained when a monoclonal antibody directed against a single plasma membrane glycoprotein was used, or when the rate of surface loss of TNP groups unoccupied by antibodies was measured. Within 15 min at 37 °C, 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 1 2 ~5 min ). Most of the intracellular radioactivity was associated with a membrane fraction of density similar to that of the plasma membrane. Over a 4-h period, there was no significant entry of labeled molecules into lysosomes. By contrast, the fluid-phase marker, horseradish peroxidase, became associated with the lysosomes within 1 h. Our results are consistent with the view that the majority of plasma membrane glycoproteins are continuously being internalized and recycled at a high rate.