Topology of nucleotide-sugar:dolichyl phosphate glycosyltransferases involved in the dolichol pathway for protein glycosylation in native rat liver microsomes.
Activities of nucleotide-sugar:dolichyl phosphate glycosyltransferases (UDP-N-acetylglucosamine:dolichyl phosphate N-acetylglucosaminyl 1-phosphotransferase, UDP-glucose:dolichyl phosphate glucosyltransferase and GDP-mannose:dolichyl phosphate mannosyltransferase) are not fully expressed in native microsomes and can be enhanced by pretreatment of the microsomes with detergent. To examine whether the latency of dolichyl phosphate glycosyltransferases in native microsomes reflects a lumenal orientation of the catalytic centre, we examined the effect of proteinase treatment of native microsomes on enzymic activity and investigated the relationship between enzymic activity and alteration of the permeability of the microsomal membrane barrier. The enzymic activities catalysing transfer of N-acetylglucosamine and glucose to lipid acceptors were proteinase-sensitive in native sealed microsomes. When various detergents were used to disrupt the membrane barrier, we found no relationship between activity of dolichyl phosphate glycosyltransferases and the latency of mannose-6-phosphatase, which is a marker of the permeability properties of the microsomal membrane. Permeabilization of the endoplasmic reticulum membrane by the pore-forming Staphylococcus aureus alpha-toxin did not affect glycosyltransferase activities. These results do not support the hypothesis that latency of the transferase activities is dependent on the permeability properties of the endoplasmic-reticulum membrane. Collectively our findings can best be explained by postulating that the active centres of the transferases are cytoplasmically oriented, while activation by detergent may be conformation-dependent.
Bossuyt
X
X
Laboratory of Biological Chemistry, Katholieke Universiteit Leuven, Belgium.
Blanckaert
N
N
D000818:Animals; D050260:Carbohydrate Metabolism; D002463:Cell Membrane Permeability; D004286:Dolichols / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism; D006031:Glycosylation; D016695:Glycosyltransferases / Q000378:metabolism*; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D051381:Rats; D013395:Sucrose / Q000378:metabolism; D010738:Type C Phospholipases / Q000494:pharmacology
D004286:Dolichols; D009711:Nucleotides; D013395:Sucrose; D016695:Glycosyltransferases; C023553:mannose-6-phosphatase; D010744:Phosphoric Monoester Hydrolases; D010738:Type C Phospholipases; D010450:Endopeptidases