Variants of the carboxyl-terminal KDEL sequence direct intracellular retention.
Soluble proteins which reside in the lumen of the endoplasmic reticulum share a common carboxyl-terminal tetrapeptide Lys-Asp-Glu-Leu (KDEL). Addition of the tetrapeptide to a normally secreted protein is both necessary and sufficient to cause retention in the endoplasmic reticulum. In order 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 expressed in the AtT-20 cell line. AtT-20 cells, a mouse anterior pituitary corticotrope cell line, synthesize, process, and secrete the pro-ACTH/endorphin precursor. Since post-translational processing in AtT-20 cells has been extensively characterized, it provides a model system in which the processing of a foreign peptide precursor (pro-NPY) and the endogenous precursor (pro-ACTH/endorphin) can be compared. Altered cDNAs encoding pro-NPY with KDEL, DKEL, RDEL, KNEL, KDQL, or KDEA at the COOH terminus were used to generate stable AtT-20 cell lines. The processing of pro-NPY to neuropeptide Y and the carboxyl-terminal peptide was studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, tryptic peptide mapping, and radiosequencing. Addition of the tetrapeptides KDEL, DKEL, RDEL, or KNEL to the COOH terminus of the neuropeptide Y precursor, a peptide hormone normally processed and secreted from neuronal cells, caused complete intracellular retention of the unprocessed prohormone in AtT-20 cells. However, KDQL and KDEA-extended pro-NPY molecules were processed and secreted like wild-type pro-NPY when expressed in AtT-20 cells. The secretion of proNPY-derived peptides in these cell lines paralleled secretion of endogenous pro-ACTH/endorphin-derived products under both basal and stimulated conditions. These mutagenesis studies demonstrate that variants of the KDEL retention signal can direct intracellular retention.
Andres
D A
DA
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Dickerson
I M
IM
Dixon
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D014644:Genetic Variation; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009478:Neuropeptide Y / Q000235:genetics; D010446:Peptide Fragments / Q000032:analysis / Q000235:genetics; D010449:Peptide Mapping; D010957:Plasmids; D011333:Pro-Opiomelanocortin / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014357:Trypsin
D009478:Neuropeptide Y; D010446:Peptide Fragments; D011498:Protein Precursors; C049531:pro-opiomelanocortin joining peptide(77-97); D011333:Pro-Opiomelanocortin; C042084:preproneuropeptide Y; D014357:Trypsin