Posttranslational processing of alpha-, beta-, and gamma-preprotachykinins. Cell-free translation and early posttranslational processing events.
We have used an in vitro transcription-translation system to study initial protein processing events of the rat substance P/neurokinin A gene products. cDNA clones for three different mRNA species, which are derived by differential RNA splicing, were subcloned into a plasmid, pGEM1, which contains the promoter for the bacteriophage SP6 RNA polymerase. In vitro synthesized mRNAs for alpha-, beta-, and gamma-preprotachykinin were translated in a wheat germ or rabbit reticulocyte cell-free system. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the translated protein products migrate consistent with the deduced molecular masses of alpha (13,035 Da)-, beta (15,003 Da)-, and gamma (13,343 Da)-preprotachykinin. The addition of dog pancreatic microsomal membranes to either cell-free translation system causes the production of a protease-resistant form of each of the three preprotachykinins which migrates with an apparent increase in molecular mass of approximately 2,000 Da. Each of these modified preprotachykinins lacks the putative signal peptide of the prepro- form, with signal peptidase cleavage occurring after the alanine residue at position 19. Both the prepro- and proforms of each tachykinin precursor molecule are recognized by antiserum R-140, an antiserum specific for the mid-portion of the undecapeptide substance P. The most likely explanation for the apparent increase in molecular mass is anomalous electrophoretic migration, since beta-preprotachykinin mRNA lacking the signal peptide encoding sequence is translated, in the absence of microsomal membranes, into a protein with the same apparent molecular mass as the modified form of beta-preprotachykinin. Therefore, each of the three preprotachykinin mRNAs are translatable, and their products are targeted to the secretory pathway by the presence of a cleavable signal peptide.
D000818:Animals; D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D009154:Mutation; D010944:Plants / Q000378:metabolism; D010957:Plasmids; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D051381:Rats; D012156:Reticulocytes / Q000378:metabolism; D015320:Tachykinins / Q000235:genetics*; D014158:Transcription, Genetic; D014908:Triticum / Q000378:metabolism
D011498:Protein Precursors; D012333:RNA, Messenger; D015320:Tachykinins; C044206:preprotachykinin