Site-directed mutagenesis and expression of PC2 in microinjected Xenopus oocytes.
The biosynthesis and post-translational maturation of PC2, a neuroendocrine-specific Kex2-like endoprotease, following expression in Xenopus oocytes is described. The initial translation product was a 75-kDa membrane-associated protein which was released from the oocytes as a glycosylated 71-kDa protein. During extended chase periods, the extracellular 71-kDa protein was converted to a mature 68-kDa product. A deletion mutant lacking a putative COOH-terminal amphipathic helix was still membrane-associated, suggesting that this domain was not essential for attachment of PC2 to membranes. Two putative proregion cleavage site mutants were also constructed. Conversion of the 75-kDa peptide to the 71-kDa peptide involved cleavage at the sequence Lys-Arg-Arg-Arg (amino acids 78-81), since mutation of this sequence to Lys-Val-Arg-Leu resulted in the secretion of the 75-kDa peptide. Extracellular conversion of the 71-kDa peptide to the 68-kDa peptide involved cleavage at the sequence Arg-Lys-Lys-Arg (amino acids 106-109), since deletion of this tetrabasic sequence resulted in secretion of the 71-kDa peptide without further conversion to the 68-kDa form. Finally, a mutation which changed a catalytically important Asp to Asn did not affect processing of proPC2. These results may be relevant to our understanding of mechanisms in the intracellular sorting and maturation of proPC2 in neuroendocrine cells.
Shennan
K I
KI
Department of Medicine, University of Birmingham, Queen Elizabeth Hospital, United Kingdom.
Seal
A J
AJ
Smeekens
S P
SP
Steiner
D F
DF
Docherty
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000502:physiology; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008845:Microinjections; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009865:Oocytes / Q000201:enzymology / Q000502:physiology*; D010957:Plasmids; D045706:Proprotein Convertase 2; D011480:Protease Inhibitors / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012333:RNA, Messenger / Q000008:administration & dosage / Q000235:genetics; D015183:Restriction Mapping; D012697:Serine Endopeptidases / Q000235:genetics*; D014158:Transcription, Genetic; D014982:Xenopus laevis
D004792:Enzyme Precursors; D009838:Oligodeoxyribonucleotides; D011480:Protease Inhibitors; D012333:RNA, Messenger; D012697:Serine Endopeptidases; D045706:Proprotein Convertase 2