Proteolytic processing of a secreted glycoprotein and O-glycosylation of mannoproteins are affected in the N-glycosylation mutant Saccharomyces cerevisiae ldb1

• Published in: Biochimica et biophysica acta Vol. 1380; no. 3; pp. 320 - 328
• Main Authors: Mañas, Paula, Olivero, Isabel, Hernández, Luis M
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 08.05.1998
• Subjects: beta-Glucosidase - genetics; beta-Glucosidase - metabolism; Carbohydrate Conformation; Glucan 1,3-beta-Glucosidase; Glycoproteins - genetics; Glycoproteins - metabolism; Glycosylation; Golgi Apparatus - genetics; Golgi Apparatus - metabolism; Isoenzymes - chemistry; Isoenzymes - genetics; Isoenzymes - metabolism; ldb1 mutant; Mannoprotein; Membrane Glycoproteins - genetics; Membrane Glycoproteins - metabolism; Mutation; N-glycosylation; O-glycosylation; Oligosaccharides - analysis; Oligosaccharides - genetics; Protein Processing, Post-Translational - genetics; Proteolytic processing; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; DPAP A, dipeptidyl aminopeptidase A; TLC, thin layer chromatography; TGN, trans-Golgi network; O-glycosylation; p-NPG, p-nitrophenyl- β- d-glucopyranoside; Mannoprotein; N-glycosylation; Proteolytic processing; endo H, endo- β- N-acetylglucosaminidase H; ldb1 mutant; PAGE, polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; ER, endoplasmic reticulum
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/S0304-4165(97)00160-8

In a previous work [P.I. Mañas, I. Olivero, M. Avalos, L.M. Hernández, Glycobiology, 7 (1997) 487–497], we described the isolation and characterization of the Saccharomyces cerevisiae ldb1 mutant which is affected in several steps of the N-glycosylation of mannoproteins probably due to a malfunction of the Golgi apparatus. Here, we found that two further functions assigned to the Golgi cisternae are also affected in the mutant: proteolytic processing of a secreted protein and O-glycosylation. We found that around 70% of the exoglucanase activity that is secreted into the culture medium by ldb1 bears an extra tetrapeptide in its NH 2-terminus due to incomplete proteolytic processing. The O-linked oligosaccharides from ldb1 mnn1 were indistinguishable from those synthesized by the parental strain mnn1. However, when the O-oligosaccharides from the wild type and ldb1 were compared, we found a significant decrease in the tetrasaccharide in the latter, as well as a concomitant increase in the disaccharide, suggesting a defect in the Kre2p/Mnt1p involved in the transfer of the third mannose of these residues.