Characterization of the carboxyl-terminal sequences responsible for protein retention in the endoplasmic reticulum.
The COOH-terminal sequence KDEL has been shown to be essential for the retention of several proteins in the lumen of the endoplasmic reticulum (Munro S., and Pelham, H. R. B. (1987) Cell 48, 899-907; Pelham, H. R. B. (1988) EMBO J. 7, 913-918; Mazzarella; R. A., Srinivasan, M., Haugejorden, S. M., and Green, M. (1990) J. Biol. Chem. 265, 1092-1101). We have previously demonstrated that variants to the KDEL retention signal, particularly at the initial two positions of the tetrapeptide, can be made without affecting its ability to direct intracellular retention when appended to the neuropeptide Y precursor (pro-NPY) (Andres, D. A., Dickerson, I. M., and Dixon, J. E. (1990) J. Biol. Chem. 265, 5952-5955). To further investigate the nature of the KDEL retention signal, oligonucleotide-directed mutagenesis and transfection was used to generate stable mouse anterior pituitary AtT-20 cell lines expressing pro-NPY mutants with variants of the KDEL sequence added to their direct carboxyl terminus. Analyses of dibasic processing and indirect immunofluorescent microscopy of AtT-20 subclones were consistent with the retention of the pro-NPY mutants bearing the COOH-terminal extensions QDEL, KEDL, or KDEI within the endoplasmic reticulum. A change in the final amino acid of the tetrapeptide from Leu to Val abolished retention completely, and the peptide hormone was processed and secreted. These results indicate that only a limited number of conservative changes can be made to the final two positions of the tetrapeptide without abolishing activity and suggest a highly specific interaction of the retention signal and the KDEL receptor.
Andres
D A
DA
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Rhodes
J D
JD
Meisel
R L
RL
Dixon
J E
JE
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009478:Neuropeptide Y / Q000378:metabolism*; D010957:Plasmids; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D018000:Receptors, Peptide; D014162:Transfection
C064459:KDEL receptor; D009478:Neuropeptide Y; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; D004247:DNA