Rapid cleavage of the endogenous PC3 prosegment and slow conversion to 74 kDa and 66 kDa proteins in AtT-20 cells.
AtT-20 cells synthesize 87 kDa and 66 kDa forms of the prohormone convertase PC3 (also known as PC1). In the present work, using biosynthetic labeling experiments (performed both at 20 degrees C and at 37 degrees C), followed by immunoprecipitation with aminoterminally and carboxyterminally-directed antisera, we have found that the first PC3 translational product was a 94 kDa protein that was then converted to an 84 kDa form. This processing was extremely rapid, occurring with a half-life of less than 2 min at 20 degrees C. The 84 kDa form was endoglycosidase H-sensitive, indicating a lack of acquisition of sugar transferred in the medial golgi. Dithiothreitol, a reducing agent that prevents the disulfide bond formation of newly synthesized proteins in the endoplasmic reticulum (ER), inhibited the processing of the 94 kDa to the 84 kDa form. However, brefeldin A (BFA), an inhibitor of ER/golgi transport, and monensin, an inhibitor of the medial/trans-golgi transport, did not affect the cleavage of the 94 kDa to the 84 kDa protein. The 84 kDa protein was converted to an endoglycosidase H-resistant form of 87 kDa that was sequentially processed to 74 kDa and 66 kDa proteins. The 87 kDa protein was immunoprecipitated by the PC3 aminoterminally and carboxyterminally-directed antisera, while the 74 kDa and 66 kDa protein were only detected with the aminoterminally-directed antibody. Radiosequencing of the 87 kDa and 66 kDa proteins indicated that the biosynthesis of the 87 kDa proteins involves the removal of the 83 amino acid prosegment, and that the processing of the 87 kDa to 66 kDa form occurred by cleavage at the carboxyterminal portion. BFA and monensin effectively interrupted the processing of the 84-87 kDa protein to the 74 and 66 kDa species. In addition, while the 84-87 kDa protein produced in monensin-treated cells was still sensitive to endoglycosidase H, the 66 kDa protein was resistant to this enzyme. These results indicate that the post-translational processing of PC3 occurs in three steps: (1) rapid conversion, probably in the ER, of the 94 kDa precursor to the 84 kDa protein by removal of the aminoterminus prosegment; (2) cleavage of the 87 kDa protein to an intermediate product of 74 kDa; and (3) production of the 66 kDa protein. The second and third steps occur in late cellular compartments such as the trans-golgi network or secretory granules and involve sequential cleavages at the carboxyterminus.(ABSTRACT TRUNCATED AT 400 WORDS)
Vindrola
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Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70112.
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000187:drug effects / Q000378:metabolism*; D020126:Brefeldin A; D002460:Cell Line; D003517:Cyclopentanes / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D006596:Hexosaminidases / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D008985:Monensin / Q000494:pharmacology; D011233:Precipitin Tests; D043484:Proprotein Convertases; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D013997:Time Factors; D014415:Tunicamycin / Q000494:pharmacology
D003517:Cyclopentanes; D014415:Tunicamycin; D020126:Brefeldin A; D008985:Monensin; D006596:Hexosaminidases; D043484:Proprotein Convertases; D016282:Aspartic Acid Endopeptidases; D004229:Dithiothreitol