Endolysosomal transport of newly-synthesized cathepsin D in a sucrose model of lysosomal storage.
Newly-synthesized soluble lysosomal enzymes are transported from the trans-Golgi network to lysosomes by a mannose 6-phosphate receptor-mediated pathway. Lysosomal storage of indigestible material has been reported to perturb the biosynthesis and the fate of lysosomal hydrolases. In this study, we have focused our attention on the last steps in the transport of newly-synthesized cathepsin D to lysosomes in sucrose-treated WI-38 fibroblasts. Pulse-chase experiments indicate that, in sucrose-treated cells, cathepsin D maturation is delayed by 2 to 4 h. By subcellular fractionation, we show that newly-synthesized cathepsin D precursors transit through organelles endowed with a high sedimentation coefficient. These organelles are recovered in the dense region of a self-forming Percoll density gradient while the bulk of hydrolytic activities is redistributed to the low density region. Only later, are the precursors delivered to organelles containing the bulk of active hydrolases. There, procathepsin D is proteolytically processed into its 31 kDa-mature form. Our results suggest that when sucrose is present, the delayed maturation of procathepsin D is related to the delivery of the polypeptides into an organelle behaving in centrifugation like lysosomes but which is poorly efficient in proteolytic processing of procathepsin D. This low proteolytic activity of this organelle could be due to its poor ability to interact with hydrolase-containing structures.
Hamer
Isabelle
I
UR(Phi)M, Laboratory of Physiological Chemistry, Facultés Universitaires N-D de la Paix, Rue de Bruxelles, 61, B-5000 NAMUR, Belgium. isabelle.hamer@fundp.ac.be
Jadot
Michel
M
D015415:Biomarkers; D002402:Cathepsin D / Q000096:biosynthesis / Q000378:metabolism*; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D003594:Cytoplasmic Granules / Q000201:enzymology; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000378:metabolism*; D005347:Fibroblasts / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D006867:Hydrolases / Q000378:metabolism; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism* / Q000494:pharmacology
D015415:Biomarkers; D004792:Enzyme Precursors; D013395:Sucrose; D006867:Hydrolases; D002402:Cathepsin D