Cathepsin D associates with lysosomal membranous protein.
The membrane-association of early biosynthetic form of cathepsin D has been demonstrated in hepatoma cells, and this membrane-association is not mediated by mannose 6-phosphate residues, implying that a mannose 6-phosphate receptor-independent mechanism operates in the sorting of cathepsin D. In this paper, to demonstrate whether cathepsin D is associated with the lysosomal membranes, an in vitro binding experiment was carried out employing lysosomal cathepsin D or microsomal procathepsin D isolated from rat liver. Immunoblotting analysis revealed that an intermediate form of cathepsin D was associated with the lysosomal membranes; this lysosomal membrane-associated cathepsin D was released from the membranes by washing with Na2CO3 (pH 10.6) but not with solutions containing mannose 6-phosphate. This suggested that cathepsin D associates with the membranes by ionic-interaction, and that the membrane-associated cathepsin D resides as a peripheral membrane protein in the lysosomal membrane fraction. To confirm that the intermediate form of cathepsin D specifically interacts with the lysosomal integral membrane proteins, the lysosomal membrane fraction was treated with trypsin and the binding experiment was conducted. The result showed that the binding capacity of cathepsin D to the lysosomal membranes was apparently abolished and cathepsin D did not rebind to the membranes. These data suggest that the intermediate form of cathepsin D is preferentially recognized by the lysosomal membranous protein which complements the mannose 6-phosphate receptor-dependent intracellular sorting mechanism.
Nishimura
Y
Y
Division of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Himeno
M
M
D000818:Animals; D002402:Cathepsin D / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015151:Immunoblotting; D008247:Lysosomes / Q000378:metabolism*; D008297:Male; D008360:Mannosephosphates / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D009997:Osmotic Pressure; D051381:Rats; D017208:Rats, Wistar; D014357:Trypsin / Q000378:metabolism
D008360:Mannosephosphates; D008565:Membrane Proteins; D014357:Trypsin; D002402:Cathepsin D