Biosynthesis and packaging of carboxypeptidase D into nascent secretory vesicles in pituitary cell lines

Metallocarboxypeptidase D (CPD) is a membrane-bound trans-Golgi network (TG N) protein. In AtT-20 cells, CPD is initially produced as a 170-kDa endogly cosidase H-sensitive glycoprotein. Within 30 min of chase, the CPD increase s to 180 kDa and is resistant to endoglycosidase H as a result of carbohydr ate maturation. CPD also undergoes an activation step required for binding to a substrate affinity resin. Blocking the protein exit from the endoplasm ic reticulum inhibits the increase in molecular mass but not the step requi red for affinity column binding, suggesting that enzyme activation precedes carbohydrate maturation and that these reactions occur in distinct intrace llular compartments. Only the higher molecular weight mature CPD enters nas cent secretory vesicles, which bud from the TGN of permeabilized AtT-20 and GH(3) cells. The budding efficiency of CPD into vesicles is 2-3-fold lower than that of endogenous proopiomelanocortin in AtT-20 cells or prolactin i n GH(3) cells. In contrast, the packaging of a truncated form of CPD, which lacks the cytoplasmic tail and transmembrane domain, was similar to that o f proopiomelanocortin, Taken together, the results support the proposal tha t CPD functions in the TGN in the processing of proteins that transit the s ecretory pathway and that the C-terminal region plays a major role in TGN r etention.