Mannose 6-phosphate-independent membrane association of cathepsin D, glucocerebrosidase, and sphingolipid-activating protein in HepG2 cells.
The membrane association of the lysosomal enzymes cathepsin D and glucocerebrosidase and its naturally occurring sphingolipid activating protein was studied in HepG2 cells. We differentially permeabilized cells with low concentrations of saponin, at which secretory proteins rinsed out completely, whereas integral membrane proteins were not released. All relevant intracellular compartments were shown to be permeabilized by saponin. Metabolic labeling showed that early precursors of cathepsin D, sphingolipid activating protein, and glucocerebrosidase were completely released from the cells, whereas more than 80% of the high molecular mass intermediates were retained by the cells. Treatment of permeabilized cells with 10 mM mannose 6-phosphate released only 50% of the cell-associated cathepsin D. Glucocerebrosidase remained membrane-associated, but cathepsin D and sphingolipid activating protein were released from the cells after proteolytic processing. Sphingolipid activating proteins and cathepsin D behaved similarly during biosynthesis and showed similar sensitivity to mannose 6-phosphate. The membrane association of the intermediate form of cathepsin D was independent of the presence of N-linked oligosaccharides. Subcellular fractionation on sucrose gradients showed that the lysosomal proteins became membrane-associated probably in the Golgi complex, and that both mannose 6-phosphate-dependent and mannose 6-phosphate-independent membrane association occur in the same compartments. We conclude that, in HepG2 cells, cathepsin D, sphingolipid activating protein, and glucocerebrosidase exhibit MPR-independent membrane association which is acquired in the same compartments beyond the rough endoplasmic reticulum.
Rijnboutt
S
S
Laboratory of Cell Biology, University of Utrecht, School of Medicine, The Netherlands.
Aerts
H M
HM
Geuze
H J
HJ
Tager
J M
JM
Strous
G J
GJ
D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002402:Cathepsin D / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005962:Glucosylceramidase / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism*; D008360:Mannosephosphates / Q000378:metabolism*; D049231:Saposins; D049128:Sphingolipid Activator Proteins; D014407:Tumor Cells, Cultured; D014415:Tunicamycin / Q000494:pharmacology
D006023:Glycoproteins; D008360:Mannosephosphates; C483345:PSAP protein, human; D049231:Saposins; D049128:Sphingolipid Activator Proteins; D014415:Tunicamycin; D005962:Glucosylceramidase; D002402:Cathepsin D