Prohormone processing in permeabilized cells: endoproteolytic cleavage of prosomatostatin in the trans-Golgi network.
Many peptide hormones are synthesized as larger precursors which undergo endoproteolytic cleavage at paired basic residues to generate a bioactive molecule. Morphological evidence has implicated either the trans-Golgi network (TGN) or immature secretory granules as the site of prohormone cleavage. To identify the site where prohormone cleavage is initiated, we have used retrovirally infected rat anterior pituitary GH3 cells which express high levels of prosomatostatin, proSRIF, (Stoller TJ, Shields D (1988) J Cell Biol 107, 2087-2095). By incubating these cells at 20 degrees C, a temperature that prevents exit from the Golgi apparatus, proSRIF accumulated quantitatively in the TGN and no proteolytic processing was evident. Following the 20 degrees C block, the cells were permeabilized and proSRIF processing determined. Cleavage of proSRIF to the mature hormone was approximately 35-50% efficient, required incubation at 37 degrees C and ATP hydrolysis, but was independent of GTP or cytosol. The in vitro ATP-dependent proSRIF processing was inhibited by inclusion of chloroquine, a weak base, CCCP, a protonophore, or by pre-incubating the permeabilized cells with low concentrations of N-ethylmaleimide or bafilomycin, both inhibitors of vacuolar-type ATP-dependent proton pumps. These data suggest that ATP is required for generation of an acidic pH in the lumen of the TGN which is necessary for the activity of prohormone processing enzymes. By exploiting a permeabilized cell system, we have demonstrated that proSRIF cleavage is initiated in the TGN, in a reaction which is facilitated by a Golgi-associated vacuolar type ATPase.
Xu
H
H
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York, NY 10461.
Shields
D
D
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002460:Cell Line; D002463:Cell Membrane Permeability; D002738:Chloroquine / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D006056:Golgi Apparatus / Q000378:metabolism*; D007700:Kinetics; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D051381:Rats; D013004:Somatostatin / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D011498:Protein Precursors; C022571:adenosine 5'-O-(3-thiotriphosphate); D013004:Somatostatin; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; C028456:prosomatostatin; D002738:Chloroquine; D000255:Adenosine Triphosphate; D010450:Endopeptidases; D006180:Proton-Translocating ATPases; D003545:Cysteine; D005033:Ethylmaleimide