In vitro mutagenesis of trypsinogen: role of the amino terminus in intracellular protein targeting to secretory granules.
The mouse anterior pituitary tumor cell line, AtT-20, targets secretory proteins into two distinct intracellular pathways. When the DNA that encodes trypsinogen is introduced into AtT-20 cells, the protein is sorted into the regulated secretory pathway as efficiently as the endogenous peptide hormone ACTH. In this study we have used double-label immunoelectron microscopy to demonstrate that trypsinogen colocalizes in the same secretory granules as ACTH. In vitro mutagenesis was used to test whether the information for targeting trypsinogen to the secretory granules resides at the amino (NH2) terminus of the protein. Mutations were made in the DNA that encodes trypsinogen, and the mutant proteins were expressed in AtT-20 cells to determine whether intracellular targeting could be altered. Replacing the trypsinogen signal peptide with that of the kappa-immunoglobulin light chain, a constitutively secreted protein, does not alter targeting to the regulated secretory pathway. In addition, deletion of the NH2-terminal "pro" sequence of trypsinogen has virtually no effect on protein targeting. However, this deletion does affect the signal peptidase cleavage site, and as a result the enzymatic activity of the truncated trypsin protein is abolished. We conclude that neither the signal peptide nor the 12 NH2-terminal amino acids of trypsinogen are essential for sorting to the regulated secretory pathway of AtT-20 cells.
D000324:Adrenocorticotropic Hormone / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003594:Cytoplasmic Granules / Q000378:metabolism* / Q000648:ultrastructure; D004274:DNA, Recombinant / Q000378:metabolism; D051379:Mice; D009154:Mutation; D010911:Pituitary Neoplasms; D011499:Protein Processing, Post-Translational; D014170:Transformation, Genetic; D014362:Trypsinogen / Q000235:genetics* / Q000378:metabolism
D004274:DNA, Recombinant; D014362:Trypsinogen; D000324:Adrenocorticotropic Hormone