Processing and routing of a membrane-anchored form of proneuropeptide Y

To investigate factors governing proteolytic processing and routing of biologically active peptides in the secretory pathway, cDNAs for preproneuropeptide Y (preproNPY) and preproneuropeptide Y fused to a membrane anchor were transfected into pituitary cells. The anchor was the transmembrane and COOH-terminal cytoplasmic domain of peptidylglycine alpha-amidating monooxygenase (PAM); these domains are essential for correct routing of integral membrane forms of PAM. Like proneuropeptide Y (proNPY), the integral membrane form of proNPY was a good substrate for the endogenous prohormone convertases, yielding soluble NPY stored in regulated secretory granules. Tethering of proNPY to the membrane resulted in only a small delay in the rate of cleavage to produce mature NPY and in the arrival of NPY in regulated secretory granules. In contrast, the COOH-terminal region of proNPY remained attached to the transmembrane/COOH-terminal domain of PAM and was rerouted to the vicinity of the trans-Golgi network, where integral membrane forms of PAM are concentrated. Thus, the COOH-terminal of proNPY cannot override the signals in the PAM membrane anchor.