Effect of monensin on synthesis, post-translational processing, and secretion of dopamine beta-hydroxylase from PC12 pheochromocytoma cells

Monensin was used to ascertain the location in the biosynthetic pathway where the 77,000-Mr membrane-bound subunit form of dopamine beta-hydroxylase is post-translationally converted to the 73,000-Mr soluble form. Treatment with low concentrations of monensin (less than or equal to 50 nM) completely depleted the cells of the norepinephrine and dopamine, had a small effect on protein synthesis, and enhanced post-translational processing of only dopamine beta-hydroxylase which was previously synthesized and presumably packaged into neurosecretory vesicles. At these low concentrations, exit from the Golgi apparatus did not appear to be blocked since stimulated secretion of a group of high molecular weight 35S]methionine-labeled proteins was not inhibited. Treatment with higher concentrations of monensin (200 nM) prevented the secretion of the 35S] methionine-labeled proteins normally released with a secretagogue, and also prevented the secretion of 3H] mannose-labeled proteins including dopamine beta-hydroxylase. Surprisingly, a group of lower molecular weight 35S]methionine-labeled proteins was now released from monensin-treated cells. Treatment with high concentrations of monensin (greater than or equal to 200 nM) appeared to block the secretory pathway prior to the packaging step, probably in the Golgi apparatus. If the proteins were packaged prior to monensin treatment, they were released upon stimulation with secretagogues. Monensin treatment (200 nM) enabled the post-translational processing of newly synthesized dopamine beta-hydroxylase, from the 77,000-Mr to the 73,000-Mr subunit form, to go to completion. The susceptibility of this 73,000-Mr subunit form to endoglycosidase H digestion was unaltered, suggesting that dopamine beta-hydroxylase from monensin-treated cells may have the same high mannose oligosaccharide content as native dopamine beta-hydroxylase. These experiments indicate that the post-translational processing of dopamine beta-hydroxylase occurs in the Golgi apparatus and may continue in immature granules prior to their acidification.