PROSOMATOSTATIN PROCESSING IN PERMEABILIZED CELLS - ENDOPROTEOLYTIC CLEAVAGE IS MEDIATED BY A VACUOLAR ATPASE THAT GENERATES AN ACIDIC PH IN THE TRANS-GOLGI NETWORK

To investigate the relationship between prohormone processing and sort ing of mature polypeptides into nascent secretory vesicles, we recentl y developed a permeabilized cell system that supports both these react ions (Xu, H., and Shields, D. (1993) J. Cell Biol. 122, 1169-1184). Ra t anterior pituitary GH(3) cells expressing high levels of prosomatost atin (proSRIF) were incubated at 20 degrees C; this temperature preven ted exit from the trans-Golgi network and inhibited proSRIF processing . Following the 20 degrees C block, the cells mere mechanically permea bilized and incubated at 37 degrees C, and proSRIF processing was dete rmined. Cleavage of proSRIF to the mature hormone required ATP hydroly sis and was inhibited by chloroquine, a weak base, or carbonyl cyanide m-chlorophenylhydrazone, a protonophore. This suggested that a proton gradient and/or an acidic pH facilitated by a vacuolar H+-ATPase was required for prohormone processing. We have now utilized the permeabil ized cell system in conjunction with the antibiotic bafilomycin A(1), a specific inhibitor of vacuolar H+-ATPases, to elucidate the role of acidic pH in prohormone processing. Here we report that (i) proSRIF pr ocessing was inhibited in vivo and in vitro by low concentrations of b afilomycin A(1), confirming the involvement of a vacuolar type ATPase in prohormone processing; (ii) the ATP requirement for processing coul d be circumvented in vitro by incubating permeabilized cells at acidic pH in the presence of protonophores, indicating that an acidic pH rat her than a H+ gradient is necessary for processing; and (iii) a pH of between 6 and 6.2 in the trans-Golgi network was optimal for proSRIF c leavage. We also demonstrate that prohormone convertase 2 exhibited te mperature-dependent activity in which proSRIF processing was inhibited at 20 degrees C in vitro. This result explains our previous observati on that prohormone processing is inhibited when intact cells are incub ated at 20 degrees C.