PROHORMONE PROCESSING IN THE TRANS-GOLGI NETWORK - ENDOPROTEOLYTIC CLEAVAGE OF PROSOMATOSTATIN AND FORMATION OF NASCENT SECRETORY VESICLES IN PERMEABILIZED CELLS

Many peptide hormones are synthesized as larger precursors which under go endoproteolytic cleavage at paired basic residues to generate a bio active molecule. Morphological evidence from several laboratories has implicated either the TGN or immature secretory granules as the site o f prohormone cleavage. To identify the site where prohormone cleavage is initiated, we have used retrovirally infected rat anterior pituitar y GH3 cells which express high levels of prosomatostatin (proSRIF) (St oller, T. J., and D. Shields. J Cell Biol. 1988. 107:2087-2095). By in cubating these cells at 20-degrees-C, a temperature that prevents exit from the Golgi apparatus, proSRIF accumulated quantitatively in the T GN and no proteolytic processing was evident; processing resumed upon shifting the cells back to 37-degrees-C. After the 20-degrees-C block, the cells were mechanically permeabilized and proSRIF processing dete rmined. 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 pr oSRIF processing was inhibited by inclusion of chloroquine, a weak bas e, CCCP, a protonophore, or by preincubating the permeabilized cells w ith low concentrations of N-ethylmaleimide, an inhibitor of vacuolar-t ype ATP-dependent proton pumps. These data suggest that: (a) proSRIF c leavage is initiated in the TGN, and (b) this reaction requires an aci dic pH which is facilitated by a Golgi-associated vacuolar-type ATPase . A characteristic feature of polypeptide hormone-producing cells is t heir ability to store the mature hormone in dense core secretory granu les. To investigate the mechanism of protein sorting to secretory gran ules, the budding of nascent secretory vesicles from the TGN was deter mined. No vesicle formation occured at 20-degrees-C; in contrast, at 3 7-degrees-C, the budding of secretory vesicles was approximately 40% e fficient and was dependent on ATP, GTP, and cytosolic factors. Vesicle formation was inhibited by GTPgammaS suggesting a role for GTP-bindin g proteins in this process. Vesicle budding was dependent on cytosolic factors that were tightly membrane associated and could be removed on ly by treating the permeabilized cells with high salt. After high salt treatment, vesicle formation was dependent on added cytosol or the di alyzed salt extract. The formation of nascent secretory vesicles contr asts with prosomatostatin processing which required only ATP for effic ient cleavage. Our results demonstrate that prohormone cleavage which is initiated in the TGN, precedes vesicle formation and that processin g can be uncoupled from the generation of nascent secretory vesicles.