Fig. 11. The late endosomal intermediate form of cathepsin D accumulates in the absence of hVps34, but there is no inhibition of the early step of procathepsin D processing. (A) Control and hVps34 KD cells were seeded at 350,000 cells/dish in 10 cm dishes. Cells were labeled with 100 µCi/ml [³⁵S]methionine, then harvested after 30 minutes or chased in medium with unlabeled methionine for 4 hours. A separate control culture was incubated with 15 mM NH₄Cl during the 4 hour chase. Cathepsin D was immunoprecipitated and subjected to SDS-PAGE and fluorography. Pro, newly synthesized 51-53 kDa procathepsin D containing a propeptide; Intermediate, cathepsin D after removal of the propeptide to generate an intermediate form that migrates at 46-48 kDa; Mature, cathepsin D after cleavage to generate the mature form that contains two non-covalently linked chains of 31 kDa and 14 kDa. (B) Immunoblot analysis of endogenous cathepsin D in whole cell lysates from control and hVps34 KD cells. (C) The hVps34 KD cells were subjected to immunogold labeling with an antibody against cathepsin D. The left panel shows a lysosome heavily labeled for cathepsin D (arrow) adjacent to a larger electron-lucent vacuole. The right panel shows cathepsin D delivered to the lumen of an enlarged vacuole (black arrowhead), with a lysosome adjacent to the vacuole (white arrowhead). Bar, 0.5 µm.