Expression of mutant ELH prohormones in AtT-20 cells: the relationship between prohormone processing and sorting.
Posttranslational processing of many proteins is essential to the synthesis of fully functional molecules. The ELH (egg-laying hormone) prohormone is cleaved by endoproteases in a specific order at a variety of basic residue processing sites to produce mature peptides. The prohormone is first cleaved at a unique tetrabasic site liberating two intermediates (amino and carboxy) which are sorted to different classes of dense core vesicles in the bag cell neurons of Aplysia. When expressed in AtT-20 cells, the ELH prohormone is also first cleaved at the tetrabasic site. The amino-terminal intermediate is then sorted to the constitutive pathway, and a portion of the carboxy-terminal intermediate is sorted to the regulated pathway. Here, we use mutant constructs of the ELH prohormone expressed in AtT-20 cells to examine the relationship between prohormone processing and consequent sorting. Prohormone which has a dibasic site in place of the tetrabasic site is processed and sorted similarly to wild type. Furthermore, mutant prohormone which lacks the tetrabasic site is processed at an alternative site comprising three basic residues. In these mutant prohormones, mature ELH is still produced and stored in dense core vesicles while amino-terminal products are constitutively secreted. However, deletion of the tetrabasic and tribasic sites results in the rerouting of the amino-terminal intermediate products from the constitutive pathway to the regulated secretory pathway. Thus, in the ELH prohormone, the location of the proteolytic processing events within the secretory pathway and the order of cleavages regulate the sorting of peptide products.
Jung
L J
LJ
Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology, Stanford University, California 94305.
Kreiner
T
T
Scheller
R H
RH
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001048:Aplysia; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D007447:Invertebrate Hormones / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011233:Precipitin Tests; D011498:Protein Precursors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013997:Time Factors
D007447:Invertebrate Hormones; D011498:Protein Precursors; C026880:egg-laying hormone, Mollusca; D004247:DNA