Response of an integral granule membrane protein to changes in pH

A key feature of the regulated secretory pathway in neuroendocrine cells is lumenal pH, which decreases between trans-Golgi network and mature secreto ry granules. Because peptidylglycine a-amidating monooxygenase, (PA-ML) is one of the few membrane-spanning proteins concentrated. in secretory granul es and is a known effector of regulated secretion, we examined its sensitiv ity to pH. Based on antibody binding experiments, the noncatalytic linker r egions between the two enzymatic domains of PAM show pH-dependent conformat ional changes; these changes occur in the presence or absence of a transmem brane domain. Integral membrane PAM-1 solubilized from rat anterior pituita ry or from transfected AtT-20 cells aggregates reversibly at pH 5.5 while r etaining enzyme activity. Over 35% of the PAM-1 in anterior pituitary extra cts aggregates at pH 5.5, whereas only about 5% aggregates at pH 7.5. PAM-1 recovered from secretory granules and endosomes is highly responsive to lo w pl-l-induced aggregation, whereas PAM-1 recovered from a light, intracell ular recycling compartment is not. Mutagenesis studies indicate that a tran smembrane domain is necessary but not sufficient for low pH-induced aggrega tion and reveal a short lumenal, juxtamembrane segment that also contribute s to pH-dependent aggregation. Taken together, these results demonstrate th at several properties of membrane PAM serve as indicators of granule pH in neuroendocrine cells.