Characterization of sequences involved in mediating degradation of ornithine decarboxylase in cells and in reticulocyte lysate

Abstract

Mouse ornithine decarboxylase is a 461-amino-acid protein that is extremely labile. A set of contiguous in-frame deletions were introduced into its C-terminal hydrophilic region. The resulting mutant proteins were expressed in cos monkey cells using an expression vector based on simian virus 40 (SV40) or by in vitro translation in reticulocyte lysate. The degradation of wild-type and mutant proteins was determined in transfected cos cells and in a degradation system based on reticulocyte lysate. Deletion mutants lacking segments of the C-terminus (amino acids 423-461, 423-435, 436-449 and 449-461) were converted into stable proteins in both experimental systems. The mutant lacking amino acids 295-309 was significantly stabilized in transfected cos cells, but was rapidly degraded in reticulocyte-lysate-based degradation mix. Our results suggest that the carboxyl-terminal region encompassing amino acids 423-461 and perhaps also amino acids 295-309 may constitute a signal recognized by the proteolytic machinery that degrades ornithine decarboxylase.