Processing of human cathepsin D in lysosomes in vitro.
The proteolytic maturation of cathepsin D polypeptides was studied in lysosomes isolated from metabolically labeled fibroblasts. In lysosomes isolated from fibroblasts labeled with [35S]methionine, 70-95% of labeled cathepsin D polypeptides were represented by a Mr = 47,000 polypeptide after a 20-min pulse and 75-min chase. When these lysosomes were incubated in vitro, up to 70% of the Mr = 47,000 polypeptide was processed to mature cathepsin D polypeptides. The processing was dependent on the integrity of the lysosomes, had an optimum between pH 6 and 7, and could be stimulated by dithiothreitol and ATP. The noncleavable ATP analogue, adenosine 5'-(beta, gamma-imido)triphosphate, and GTP, CTP, and UTP could not substitute for ATP. The ATP-dependent stimulation was associated with an acidification of lysosomes. It was inhibited by agents that dissipate the lysosomal pH gradient (carbonyl cyanide p-trifluoromethoxyphenylhydrazone, N,N'-dicyclohexylcarbodiimide, nigericin, NH4Cl). A stimulatory effect of ATP was observed also at pH 5.5. The stimulation at pH 5.5 was not associated with acidification of lysosomes and was resistant to protonophores. Inhibitors of lysosomal cysteine proteinases and N-ethylmaleimide inhibited the processing. In the presence of ATP the processing activity was partially protected from inhibition by N-ethylmaleimide. In conclusion, the maturation of cathepsin D in lysosomes depends on cysteine proteinases and is stimulated by the ATP-driven acidification of lysosomes. In addition, ATP stimulates maturation at pH 5.5 by a mechanism not involving the proton pump.
D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002402:Cathepsin D / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000378:metabolism*; D008970:Molecular Weight; D009550:Nigericin / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D014634:Valinomycin / Q000494:pharmacology
D011480:Protease Inhibitors; D014634:Valinomycin; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D002402:Cathepsin D; D005033:Ethylmaleimide; D009550:Nigericin; D004229:Dithiothreitol