IDENTIFICATION OF ROUTING DETERMINANTS IN THE CYTOSOLIC DOMAIN OF A SECRETORY GRANULE-ASSOCIATED INTEGRAL MEMBRANE-PROTEIN

We have investigated the trafficking of integral membrane peptidylglyc ine alpha-amidating monooxygenase (PAM) in the neuroendocrine AtT-20 c ell line, This bifunctional enzyme has two domains which together cata lyze the COOH-terminal alpha-amidation of peptidylglycine substrates y ielding amidated products stored in secretory granules, As soluble pro teins, both catalytic domains were independently targeted to secretory granules, In contrast, membrane PAM was largely localized to the tran s-Golgi network (TGN), Upon truncation of its cytoplasmic COOH-termina l domain, membrane PAM was less efficiently cleaved by secretory granu le enzymes and accumulated on the plasma membrane, When transferred to the lumenal domain of the interleukin 2 receptor alpha-chain (Tac pro tein), the cytoplasmic domain of PAM caused rerouting of Tac from the surface to the TGN and supported internalization of Tac antibody from the plasma membrane, To define sequences in the cytoplasmic domain of integral membrane PAM involved in its trafficking, we expressed PAM pr oteins containing truncations, deletions, or point mutations in the CO OH-terminal cytoplasmic domain. PAM proteins were not retained in the TGN when half of the cytoplasmic domain was deleted; such proteins acc umulated on the plasma membrane, were not efficiently internalized, an d were cleaved to generate a bifunctional PAM protein that was not sto red in secretory granules, A tyrosine-based internalization motif was identified, which was not required for efficient cleavage of full-leng th integral membrane PAM by secretory granule enzymes, Deletion of an 18-amino acid domain surrounding this Tyr residue both diminished clea vage of membrane PAM by secretory granule enzymes and eliminated inter nalization of PAM from the plasma membrane, The cytoplasmic domain is responsible for retaining membrane PAM in the TGN and for retrieving m embrane PAM from the cell surface, while the lumenal catalytic domains of PAM appear to be responsible for targeting the protein to secretor y granules.