Characterization of the carboxyl-terminal sequences responsible for protein retention in the endoplasmic reticulum
Abstract
The C-terminal sequence Lys-Asp-Glu-Leu (KDEL) has been shown to be essential for the retention of several soluble proteins in the lumen of the endoplasmic reticulum. Addition of the tetrapeptide to a normally secreted protein is both necessary and sufficient to cause retention in the endoplasmic reticulum (ER). To characterize the critical residues in the KDEL signal, cDNAs encoding proneuropeptide Y (pro-NPY) with the 4-amino acid carboxyl terminal extension KDEL or a series of KDEL variants were used to generate stable AtT-20 cell lines. AtT-20 cells synthesize, process, and secrete the pro-ACTH/endorphin precursor. The post-translational processing in AtT-20 cells has been extensively characterized, providing a model system in which the processing of the foreign peptide precursor (pro-NPY) and the endogenous precursor (pro-ACTH/endorphin) can be compared. The subcellular localization of these mutants was determined by indirect immunofluorescence microscopy. The processing of the pro-NPY mutants to neuropeptide Y and the carboxyl-terminal peptide was studied using sodium dodecyl-sulfate-polyacrylamide gel electrophoresis, tryptic peptide mapping, and radiosequencing. The KDEL retention signal, when appended to the COOH-terminal of wild-type pre-pro-NPY, was found to be both necessary and sufficient to cause complete ER retention of the unprocessed precursor within AtT-20 cells. Additionally, a number of mutations to the initial half of the tetrapeptide signal can be made without affecting ER localization. These data indicate that soluble proteins in the ER may carry sequences other than KDEL at their COOH terminal. These studies also emphasize the vital role of the terminal two amino acids (EL) in the recognition of the tetrapeptide by the KDEL receptor. Amino acid substitution experiments showed that the third position requires an acidic side chain amino acid while a large aliphatic amino acid is required at the fourth position, defining a partial consensus sequence.
Degree
Ph.D.
Advisors
Dixon, Purdue University.
Subject Area
Molecular biology|Biochemistry
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