Equilibration of fucosyl glycoprotein pools in HeLa cells

Abstract

The pool sizes, label equilibration times, and specific radioactivity relationships of fucosyl glycoproteins and precursors have been examined in exponentially growing HeLa S3 cells (generation time about 23 h) using a quantitative radioisotopic approach. The specific radioactivity of the precursor GDP-fucose (pool size 0.52 +/- 4% nmol/10(7) cells) equilibrates with radioactive fucose in the medium in about 1 h. 10(7) cells contain 5.3 +/- 16% nmol of glycoprotein fucose of which 96-98% resides in or on the cell surfaces and is equilibrated isotopically within 22 h of labeling; 2% or less is in an internal pool, some of which is precursor to plasma membranes and some of which is released as soluble glycoprotein directly to the medium without random mixing with the plasma membrane glycoprotein. Because we cannot rule out the presence of internal free fucose, 2% of the total glycoprotein fucose could be in the degradative pathway being recycled internally before release of free fucose. In rate terms and in a particular culture where 10(7) cells contained 4.4 nmol of glycoprotein fucose, a total of 11.1 nmol of glycoprotein fucose is synthesized per generation (9-11% of the total cell glycoprotein fucose/h). Of this, 2.5 nmol of glycoprotein fucose per generation is released directly into the growth medium without mixing with the plasma membrane glycoprotein fucose. The small internal pool feeding glycoprotein fucose to the plasma membrane does so at the rate of 8.6 nmol/10(7) cells per generation, 4.2 nmol per generation of which, after mixing with the plasma membrane glycoprotein fucose, is ultimately released into the growth medium, 75-80% as free frucose. This release process is independent of cell density and the presence of serum in the growth medium.