Abstract
Evidence for extensive subcellular organization of asparagine-linked oligosaccharide processing and lysosomal enzyme phosphorylation.
Highlights
Ated on a continuous sucrose gradient andassayed for acetylglucosamine1-phosphodiestera-N-acetylglucosaminienzymes involved in the processing of asparagine- dase’ (9, lo), and N-acetylglucosamine 1-phosphotransferase linked oligosaccharides
The data presented here indicate that the eventosf aspar- of different densities indicates that the Man-6-P recognition agine-linked oligosaccharide processing occur in multiple re- marker of lysosomal enzymes is generated intwo regions, both gions within the cell
With the exception of glucosidases I and 11, bound lysosomal enzymes contain sialylated, phosphorylated most of the enzyme activities measuredin these experiments hybrid oligosaccharides indicates that newly synthesized lyhave been shown to fractionatewith the Golgi marker, galac- sosomal enzymespass all the way through theGolgi complex tosyltransferase, in other systems [5,6,7,8,9,10]
Summary
All assays were performedwith two concentrations of protein. Product formation was proportional to protein concentration in each assay on each gradient fraction except for GlcNAc transferase IV, where lower concentrationpoints gave valuestoo low to assess linearity. Endogenous transfer in the absence of fetuin acceptor was negligible across the gradient.Values of controls lacking enzyme have been subtracted. The sucrose gradient fractions were divided into two portions with 25% used for enzyme assays and 75% used for analysis of endogenously-labeled oligosaccharides. The membrane pellets were resuspended diate in distribution between the glucosidases and galactosyltransferase, a late processingenzyme and a marker of the trans Golgi membranes [14] In this cell type the phosphodiester glycosidase was situated between the N-acetylglucoswith a Pasteur pipette in 1 ml of mM Tris, pH 7, 100 mM NaC1, 5.
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