Upregulation and storage of the lysosomal cysteine proprotease procathepsin L

The lysosomal cysteine protease cathepsin L is upregulated in Ras-transformed fibroblasts and stored as precursor procathepsin L by these cells, but the mechanism of cathepsin L upregulation has not been elucidated. In this study, the signaling pathways responsible for cathepsin L upregulation in Ras-transformed 208F rat fibroblasts and Ras-transformed rat ovarian surface epithelial (ROSE) cells were determined. The Raf pathway was necessary for full upregulation of cathepsin L in the fibroblasts, whereas both the Raf pathway and phosphatidylinositol-3-kinase (PI3K) pathway were necessary for cathepsin L upregulation in the epithelial cells. While significant accumulation of procathepsin L was observed in both Ras-expressing cell types, 208F fibroblasts secreted a larger percentage of the upregulated cathepsin L protein while the ROSE cells transported a larger percentage of the upregulated cathepsin L to lysosomes.; In virally-transformed KNIH mouse fibroblasts expressing Ras, procathepsin L accumulates in small, electron-dense vesicles and in multivesicular bodies, organelles containing small internal vesicles within a larger limiting membrane, as determined by electron microscopy. These compartments were endosomes as they could be accessed by uptake of horseradish peroxidase (HRP) and contained the endosomal marker protein CD63. As this compartment was labeled with antibodies specific to the procathepsin L propeptide region to avoid the detection of mature cathepsin L in multivesicular late endosomes, these data suggest that there are two distinct multivesicular endosomal compartments in KNIH fibroblasts.; Procathepsin L targeting was studied by ectopic expression of wildtype and mutant cathepsin L proteins in untransformed fibroblasts. Expressed wildtype cathepsin L protein accumulated inside cells as procathepsin L in a vesicle population distinct from lysosomes. A Y40A-cathepsin L protein accumulated in the TGN and was not secreted, suggesting that the cathepsin L propeptide might interact with an unidentified receptor protein to exit this compartment. A V334A-cathepsin L protein accumulated in vesicles distinct from lysosomes, similar to wildtype cathepsin L, but secretion was significantly reduced, suggesting that the cathepsin L C-terminus might interact with a receptor protein in order to be secreted by these cells.