Identification of a transferable sorting domain for the regulated pathway in the prohormone convertase PC2.
The mammalian subtilisin-like endoproteases furin and PC2 catalyze similar reactions but in different parts of the cell: furin in the trans-Golgi network and PC2 in dense-core granules. To map targeting domains within PC2, chimeras were constructed of the pro-, catalytic, and middle domains of furin with the carboxyl-terminal domain of PC2 (F-S-P) or of the pro- and catalytic domains of furin with the middle and carboxyl-terminal domains of PC2 (F-N-P). Their behavior in stable transfected AtT-20 cells was compared to a furin mutant truncated after the middle domain (F-S), wild-type furin, and with wild-type PC2. F-S-P, F-N-P, and F-S were catalytically active and underwent post-translational proteolysis and N-glycosylation with similar kinetics to wild-type furin. The truncated furin mutant was not stored intracellularly, whereas both chimeras, like PC2, showed intracellular retention and regulated release. Immunofluorescence and immuno-electron microscopy showed the presence of the chimeras and PC2 in dense-cored secretory granules together with proopiomelanocortin immunoreactivity. PC2 was sorted more efficiently than F-S-P, and the inclusion of the middle domain (F-N-P) further enhanced intracellular retention. It is concluded that sorting of PC2 into the regulated pathway depends on its carboxyl terminus. The middle domain may provide additional sorting determinants or a conformational framework for expression of the sorting signal.
Creemers
J W
JW
Department of Clinical Biochemistry, University of Cambridge, Addenbrookes Hospital, Hills Road, Cambridge CB2 2QR, United Kingdom.
Usac
E F
EF
Bright
N A
NA
Van de Loo
J W
JW
Jansen
E
E
Van de Ven
W J
WJ
Hutton
J C
JC
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D003594:Cytoplasmic Granules / Q000201:enzymology; D045683:Furin; D006031:Glycosylation; D006056:Golgi Apparatus / Q000201:enzymology; D006801:Humans; D016253:Microscopy, Immunoelectron; D016297:Mutagenesis, Site-Directed; D045706:Proprotein Convertase 2; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013381:Subtilisins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D011993:Recombinant Fusion Proteins; D013381:Subtilisins; D045683:Furin; D045706:Proprotein Convertase 2