Biosynthesis and intracellular transport of alpha-2,6-sialyltransferase in rat hepatoma cells.
We investigated biosynthesis, intracellular transport and release of beta-galactoside alpha-2,6-sialyltransferase in a dexamethasone-inducible rat hepatoma cell line. Confluent cells were induced by 10 microM dexamethasone for 24 h, and metabolically labelled with [35S]methionine/cysteine, followed by immunoprecipitation of sialyltransferase and electrophoretic/fluorographic analysis. The 35S-labelled enzyme was synthesized as a 46-kDa precursor, converted to an intermediate 47-kDa form after 1 h, and gradually to a mature form of 48 kDa within the following 3 h. By means of either tunicamycin inhibition of N-glycosylation or cleavage of N-glycans from isolated sialyltransferase using N-glycosidase F, the sizes of the precursor and the mature form were reduced to 41 kDa and 43 kDa, respectively. After a 4-h chase, treatment with endoglycosidase H revealed two distinct molecular forms of sialyltransferase, bearing either two N-acetyllactosamine-type or one oligomannose-type and one N-acetyllactosamine-type N-linked sugar chain. In addition, sialyltransferase became sensitive to neuraminidase digestion after a 4-h chase. The half-life of intracellular [35S]sialyltransferase was estimated at 3 h. A soluble form was detectable in the supernatant, 2 h after the pulse. Only 12% of the initially labelled sialyltransferase was found in the medium after 12 h, while 73% of the enzyme was degraded intracellularly. To characterize a possible intracellular degradation site, we studied intracellular transport in the presence of either secretion-blocking or acidotropic agents or protease inhibitors. Degradation was significantly delayed by all treatments. Our results show that sialyltransferase follows the secretory pathway as a membrane protein and is retained at a late Golgi stage. We suggest that the bulk of sialyltransferase in rat hepatoma cells is diverted to a post-Golgi degradation pathway. This route contrasts with the post-Golgi trafficking of beta-1,4-galactosyltransferase in HeLa cells, which is constitutively secreted [Strous, G. J. A. M. & Berger, E. G. (1982) J. Biol. Chem. 257, 7623-7628].
Bosshart
H
H
Physiologisches Institut, Universität Zürich, Switzerland.
Berger
E G
EG
D000818:Animals; D001692:Biological Transport; D003907:Dexamethasone / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006207:Half-Life; D007163:Immunosorbent Techniques; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D019158:N-Acetylneuraminic Acid; D009439:Neuraminidase / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism; D051381:Rats; D012794:Sialic Acids / Q000378:metabolism; D012799:Sialyltransferases / Q000096:biosynthesis / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D011498:Protein Precursors; D012794:Sialic Acids; D003907:Dexamethasone; D012799:Sialyltransferases; D009439:Neuraminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D019158:N-Acetylneuraminic Acid; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase