Rapid endocytosis and recycling of wheat germ agglutinin binding sites on CHO cells: evidence for two compartments in a nondegradative pathway

Abstract

The internalization and recycling of CHO cell plasma membrane components have been quantified by using iodinated wheat germ agglutinin (WGA) as an adsorptive tracer. Most of these binding sites are thought to be composed of a subpopulation of plasma membrane proteins called high-molecular-weight acidic glycoproteins (HMWAG). Greater than 90% of the WGA bound on the cell surface can be removed by brief treatment with N-acetylglucosamine (GlcNAc). At 37 degrees C, endocytosis of WGA that had been allowed to bind to the surface at 4 degrees C is curvilinear with an initial rapid phase occurring with a t1/2 of 6-8 min. Within 20 min, accumulation has slowed gradually to steady-state with 65% of the cell-associated WGA located intracellularly or resistant to removal by GlcNAc. These portions are unaffected by increasing the extracellular concentration of WGA from 0.003 microM to 2.8 microM. By using pulse-chase experiments, the observed decrease in rate of endocytosis is shown to be due to return of the WGA-HMWAG complexes to the cell surface. More than 60% of the WGA that had been internalized is recycled within 30 min, with a mean t1/2 of 17 min. Recycling involved at least two intracellular populations where a significant fraction (less than 30%) of the WGA-HMWAG complexes are lost gradually from the rapidly recycling pool. Most of the WGA-HMWAG complexes that had internalized are not delivered to the lysosome. These results demonstrate the magnitude of rapid and continuous recycling of WGA binding sites between the cell surface and endosomes in fibroblasts.