beta-Glucuronidase is transported slowly to lysosomes in BW5147 mouse lymphoma cells: evidence that the prelysosomal enzyme is not restricted to the endoplasmic reticulum.
The post-translational processing of beta-glucuronidase in BW5147 mouse lymphoma cells is slow relative to other newly synthesized lysosomal enzymes. To characterize this slow maturation the acid hydrolase was immunoprecipitated from cells pulse-labeled with [2-3H]mannose. Radiolabeled beta-glucuronidase migrated as the precursor form of the enzyme for up to 4 h of chase, whereas another acid hydrolase, beta-galactosidase, was processed completely to its mature form within this same time period. Both beta-glucuronidase and beta-galactosidase obtained high levels of mannose 6-phosphate (Man 6-P) within 60 min of their biosynthesis. The Man 6-P content of beta-galactosidase declined rapidly during a subsequent chase while that of beta-glucuronidase remained high during the first 4 h of chase and then slowly declined. 3H-Labeled phosphorylated high mannose-type oligosaccharides isolated from beta-glucuronidase after 1 h of chase were composed primarily of species with one or two phosphodiester groups, but oligosaccharides with one and two phosphomonoesters became the predominant phosphorylated species with longer chase times. The phosphorylated oligosaccharides attached to other newly synthesized acid hydrolases, on the other hand, contained primarily phosphodiester species at all chase times. When BW5147 cells were pulsed with [3H]mannose and chased in the presence of monensin to disrupt transport, the number of phosphorylated oligosaccharides recovered from beta-glucuronidase was comparable to the quantity recovered from the enzyme produced by non-drug-treated cells. The number of phosphorylated units recovered from all other newly synthesized acid hydrolases, however, was greater in the presence of the ionophore than in its absence. Nondenaturing gel electrophoresis studies indicated that beta-glucuronidase existed in two forms at steady state within BW5147 cells and, as such, was similar to liver beta-glucuronidase in which a large percentage of the enzyme was present as a complex bound to egasyn. These data suggest that newly synthesized beta-glucuronidase produced by BW5147 cells complexes with an egasyn-like protein within the endoplasmic reticulum. This interaction retards the enzyme's migration through the secretory apparatus but does not prevent its access to Golgi-associated processing enzymes.
Lazzarino
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Department of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Gabel
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CA
D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005966:Glucuronidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006020:Glycopeptides / Q000302:isolation & purification; D008223:Lymphoma / Q000201:enzymology*; D051379:Mice; D009844:Oligosaccharides / Q000302:isolation & purification; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational
D006020:Glycopeptides; D009844:Oligosaccharides; D005966:Glucuronidase