IDENTIFICATION OF SUBCELLULAR COMPARTMENTS CONTAINING PEPTIDYLGLYCINEALPHA-AMIDATING MONOOXYGENASE IN RAT ANTERIOR-PITUITARY

Both soluble and integral membrane forms of peptidylglycine alpha-amid ating monooxygenase (PAM) are expressed in the rat anterior pituitary, making it an ideal model system for studying the routing of proteins into secretory granules. To identify the subcellular compartments invo lved in the routing of integral membrane PAM, we used subcellular frac tionation, metabolic labeling and immunoblot analysis. Mature secretor y granules were found to contain full-length integral membrane PAM alo ng with a significant amount of soluble PAM generated by endoproteolyt ic cleavage. PAM proteins were not co-distributed with tyrosylprotein sulfotransferase activity during sucrose gradient centrifugation, indi cating that the trans-Golgi/TGN is not a major PAM-containing compartm ent at steady state. Fractionation of the 4,000 g and 10,000 g pellets obtained by differential centrifugation identified a significant amou nt of integral membrane PAM in a light fraction lacking soluble secret ory granule proteins. Metabolic labeling experiments with primary ante rior pituitary cells demonstrated that integral membrane PAM enters a light compartment with similar properties only after exit from the tra ns-Golgi/TGN. Comparison of the metabolic labeling and immunoblot anal yses suggests that PAM in this post-trans-Golgi/TGN compartment is in organelles involved in the intracellular recycling of integral membran e PAM. Small amounts of full-length integral membrane PAM were also re covered in fractions containing internalized transferrin and may be in an endosomal compartment following retrieval from the cell surface.