Synthesis and assembly of HeLa cell plasma membrane glycoproteins and proteins.
At least 60 percent of the fucose residues in HeLa cell glycoprotein are nonreducing, terminal, and closely proximal to the protein carbohydrate linkage. As determined by pulse-labeling with (3H) fucose and sizing glycopeptides in Sephadex chromatography these residues are added near the time of completion of oligosaccharide chains. Glycoproteins, the large bulk if not the only macromolecules labeled with radioactive fucose in HeLa cells, were not soluble in ethanol or chloroform-methanol, 2:1, but were substanially solubilized by chloroform-methanol-water, 10:10:3. Folch extraction of labeled cells and analysis of the upper phase revealed little if any (3H) fucose-labeled glycosphingolipids. Studies on the distribution of radioactively labeled glycoprotein in various cell fractions show that in uniform labeling conditions fucosylated glycoproteins accumulate in the plasma membrane specifically. Pulse-chase and protein synthesis inhibitor studies show that there is an internal pool of completed fucosylated glycoprotein, taking not less than 12 min to deplete. From this pool newly synthesized glycoprotein moves to the plasma membrane with a transit time of 12 min and little was found soluble in the cell. By contrast, a pool of protein labeled with 14C-aminoacids and precursor to plasma membrane protein is small and depleted almost immediately. From this pool newly synthesized protein molecules move to the plasma membrane with a transit time of less than 2 min. It would appear that these two distinct molecular components of plasma membranes may be assembled into membranes sequentially or into the plasma membrane independently.
D000596:Amino Acids / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D005643:Fucose / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis*; D006026:Glycoside Hydrolases; D006367:HeLa Cells / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000096:biosynthesis*; D010452:Peptide Biosynthesis; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors
D000596:Amino Acids; D006023:Glycoproteins; D009363:Neoplasm Proteins; D005643:Fucose; D003513:Cycloheximide; D006026:Glycoside Hydrolases