Expression of normal and mutant human pre-pro-insulins in Xenopus oocytes.
Conveniently situated PstI sites were used to delete a major segment from the C-peptide coding region of a human pre-pro-insulin cDNA. The resultant mutant cDNA encoded a protein with the structure: pre-peptide B chain--Arg-Arg-Glu-Ala-Glu-Asp-Leu-Gln-Lys-Arg-A chain. Normal and mutant human pre-pro-insulin cDNAs were used as templates for the synthesis of mRNA in a reaction catalysed by T7 RNA polymerase. The mRNAs were then microinjected into Xenopus oocytes to determine the effect of the deletion on the secretion of pro-insulin. When normal pre-pro-insulin mRNA was microinjected, pre-pro-insulin was processed to pro-insulin, which in turn was secreted into the media. When the mutant pre-pro-insulin mRNA was microinjected, however, mutant pro-insulin could be detected in the oocytes but at a much lower level than the normal pro-insulin. No mutant pro-insulin could be detected in the media. The stability of the mRNAs in the oocytes was investigated by microinjecting [32P]mRNA. 24 and 48 h after microinjection, the recovery of [33P]mRNA from the oocytes was 95 and 24% and 20 and 16% of that injected, for the normal and mutant mRNAs, respectively. In a cell-free translation system supplemented with dog pancreatic microsomal membranes, the pre-peptide was cleaved from the normal pre-pro-insulin but not from the mutant pre-pro-insulin. These results suggest that C-peptide plays an important role in the segregation of pro-insulin within and transport through the cellular secretory pathway.
Shennan
K I
KI
Department of Medicine, University of Birmingham, Queen Elizabeth Hospital, U.K.
Docherty
K
K
D000818:Animals; D002474:Cell-Free System; D004247:DNA / Q000235:genetics*; D004262:DNA Restriction Enzymes; D005260:Female; D005796:Genes; D006801:Humans; D007328:Insulin; D009154:Mutation; D009865:Oocytes / Q000378:metabolism*; D011384:Proinsulin / Q000235:genetics*; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic; D014982:Xenopus laevis
D007328:Insulin; D011498:Protein Precursors; D012333:RNA, Messenger; C013737:preproinsulin; D004247:DNA; D011384:Proinsulin; D004262:DNA Restriction Enzymes