Carboxy-terminal SEKDEL sequences retard but do not retain two secretory proteins in the endoplasmic reticulum.

Abstract

Abstract. The sequence Ser-Glu-Lys-Asp-Glu-Leu (SEKDEL) has been shown to be a signal which leads to retention of at least two proteins in the endoplasmic reticulum of animal cells (Munro and Pelham, 1987). In this study we tested the function of this signal by appending it to two secretory proteins, rat growth hormone and the ot subunit of human chorionic gonadotrophin (hCG-o0. We used oligonucleotidedirected mutagenesis and expression to generate proteins with SEKDEL added to the exact COOH termini and then carried out a detailed analysis of their transport in monkey COS cells. We found that transport was not blocked for either protein, but rather that the half-time for secretion was increased about sixfold for both proteins. Analysis of oligosaccharide processing on hCG-ot-SEKDEL and indirect immunofluorescence microscopy on cells expressing both proteins was consistent with a retardation of transport between the endoplasmic reticulum and the Golgi apparatus. A change in the last amino acid of the SEKDEL sequence from Leu to Val abolished the retardation almost completely, suggesting a highly specific interaction of the sequence with a receptor. A change in the first amino acid had little or no effect on retardation. We conclude that the SEKDEL signal can have strong effects on reducing the rate of protein exit from the endoplasmic reticulum without generating absolute retention. Presumably other features of protein structure must be important to generate absolute retention.M ECHANISMS exist for sorting of proteins within the exocytic pathway of eucaryotic cells. This sorting leads to retention of specific proteins within the ER and subcompartments of the Golgi apparatus, and is thought to be mediated by sorting signals in the proteins themselves and receptors in the pathway (reviewed by Rose and Doms, 1988). One of the simplest and clearest examples of a sorting signal is the tetrapeptide sequence Lys-Asp-Glu-Leu (KDEL) which Munro and Pelham (1987) noted at the COOH termini of four proteins that are residents of the ER. Deletion of this sequence from one of these resident ER proteins, GRP78, resuited in slow secretion of the truncated GRP78, and addition of the SEKDEL sequence from GRP78 to the secretory protein lysozyme resulted in retention of lysozyme in the ER. Although the receptor for the KDEL sequence has not yet been identified, it was suggested that it might reside in an early Golgi compartment and function through a recycling process that retrieves resident ER proteins back from the Golgi apparatus. Evidence for such a mechanism has been obtained both in mammalian cells (Pelham, 1988) and in yeast which use the related retention signal HDEL .Because the function of the KDEL signal had only been tested on one secretory protein from mammalian cells, we decided to test it further by appending it to two well-characterized secretory proteins that were under study in our laboratory, rat growth hormone (rGH), 1 and the ot subunit of human chorionic gonadotropin (hCG-o0. rGH is a nonglycosylated monomeric...

Expression and Partial Purification of a Recombinant Secretory Form of Human Liver Carboxylesterase

“…The present study shows the conversion of a membrane-bound form of a human liver carboxylesterase to a secretory enzyme by changing its C-terminal leucine to an arginine residue. In agreement with previous studies on KDEL-tagged proteins (30,39), substitution of the C-terminal leucine of a fully mature human liver carboxylesterase disrupts selective retrieval to the endoplasmic reticulum by the KDEL sorting receptor. Expression of the mutated carboxylesterase cDNA (pRc/CMV-mhCaE) was compared to expression of the normal cDNA (pRc/CMV-hCaE) stably integrated into the same cell line.…”

Human homologue of murine tumor rejection antigen gp96: 5'-regulatory and coding regions and relationship to stress-induced proteins.

“…Presumably, a fraction ofgp96 makes its way to the cell surface either through the continuity of endoplasmic reticulum membrane with the plasma membrane or by a specific mechanism that modulates the influence of the KDEL sequence. Some normally secreted proteins-e.g., rat growth hormone and a subunit of chorionic gonadotropin-are not retained in the endoplasmic reticulum in spite of the addition of a C-terminal KDEL sequence, supporting the notion that KDELmediated endoplasmic reticular retention of gp96 is not absolute (35).…”

CBP-140, a Novel Endoplasmic Reticulum Resident Ca2+-binding Protein with a Carboxy-terminal NDEL Sequence Showed Partial Homology with 70-kDa Heat Shock Protein (hsp70).

“…On the other hand, Ozawa and Muramatsu (25) showed that a mutant reticulocalbin lacking HDELwas secreted in the culture medium. These observations are not so contradictory since the efficiency of retention can vary, depending on the proteins being analyzed (36). Thus we can propose the NDELas ER retention signal for CBP-140 though none of the reported signals has yet included it.…”