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

Abstract

Metallocarboxypeptidase D (CPD) is a membrane-bound trans. Golgi network (TGN) protein. In AtT-20 cells, CPD is initially produced as a 170-kDa endoglycosidase H-sensitive glycoprotein. Within 30 min of chase, the CPD increases to 180 kDa and is resistant to endoglycosidase H as a result of carbohydrate maturation. CPD also undergoes an activation step required for binding to a substrate affinity resin. Blocking the protein exit from the endoplasmic reticulum inhibits the increase in molecular mass but not the step required for affinity column binding, suggesting that enzyme activation precedes carbohydrate maturation and that these reactions occur in distinct intracellular compartments. Only the higher molecular weight mature CPD enters nascent secretory vesicles, which bud from the TGN of permeabilized AtT-20 and GH3 cells. The budding efficiency of CPD into vesicles is 2-3- fold lower than that of endogenous proopiomelanocortin in ART-20 cells or prolactin in GH3 cells. In contrast, the packaging of a truncated form of CPD, which lacks the cytoplasmic tall and transmembrane domain, was similar to that of proopiomelanocortin. Taken together, the results support the proposal that CPD functions in the TGN in the processing of proteins that transit the secretory pathway and that the C-terminal region plays a major role in TGN retention.