Post-translational protein modification in the endoplasmic reticulum. Demonstration of fatty acylase and deoxymannojirimycin-sensitive alpha-mannosidase activities.
We have previously described a hybrid protein, GHHA, that contains a fragment of the influenza hemagglutinin joined to the C terminus of a nearly complete rat growth hormone (Rizzolo, L.J., Finidori, J., Gonzalez, A., Arpin, M., Ivanov, I.E., Adesnik, M., and Sabatini, D.D. (1985) J. Cell Biol. 101, 1351-1362). GHHA was transported from the rough endoplasmic reticulum (ER) to a smooth cisterna, continuous with the rough ER, but proximal to the Golgi apparatus. We have now labeled GHHA with [3H]palmitate, demonstrating that fatty acylation can occur in the ER. As expected for a thioester linkage, the label was released from GHHA by hydroxylamine and identified as palmitic acid by thin-layer chromatography. In a second study, we analyzed the structure of the N-linked carbohydrate chain of GHHA. The N-linked oligosaccharides, all high-mannose type, were released by endoglycosidase H and size-fractionated by high pressure liquid chromatography. The predominant structures were Glc1Man8GlcNAc and Man8GlcNAc, indicating that only 2 or 3 glucose and 1 mannose residues were removed from the original Glc3Man9GlcNAc2. Determination of the structure by acetolysis fragmentation indicated that a single Man8GlcNAc isomer was formed by a deoxymannojirimycin-sensitive alpha-mannosidase. This contrasts with a previously characterized ER alpha-mannosidase (Bischoff, J., Liscum, L., and Kornfeld, R. (1986) J. Biol. Chem. 261, 4766-4774) that generates the same isomer, but is deoxymannojirimycin-resistant. These data suggest the possibility that different enzymes are partitioned within the ER.
Rizzolo
L J
LJ
Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322.
Kornfeld
R
R
D017485:1-Deoxynojirimycin; D000123:Acetyltransferases / Q000378:metabolism*; D000215:Acylation; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005796:Genes; D005944:Glucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D008361:Mannosidases / Q000235:genetics* / Q000378:metabolism; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D014158:Transcription, Genetic; D043323:alpha-Mannosidase
D000900:Anti-Bacterial Agents; D010169:Palmitic Acids; D017485:1-Deoxynojirimycin; D019308:Palmitic Acid; D000123:Acetyltransferases; D008361:Mannosidases; D043323:alpha-Mannosidase; D005944:Glucosamine