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Introduction

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Figure 1  
The secretory pathway. Proteins move from the endoplasmic reticulum through the Golgi apparatus to the cell surface. Transport of secretory and plasma membrane proteins is essentially unidirectional, and in most cases their concentration increases as they pass along the pathway. Retrograde transport (red arrow) allows proteins not destined for secretion to be retrieved from the Golgi apparatus and returned to the ER.

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Proteins that are secreted from eukaryotic cells are inserted into the endoplasmic reticulum (ER), and transported to the Golgi apparatus and to the cell surface, where they are released, as shown in the general model of Figure 1 (see Protein trafficking). In the mid-1980s, it was generally accepted that transport of cargo proteins along this pathway is mediated by membrane-bound transport vesicles that bud from one compartment and fuse with another.

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A paradigm for how vesicle-mediated transport might work was provided by the spectacular advances in understanding receptor-mediated endocytosis. The principle is that the transmembrane receptors that recognize extracellular ligands contain in their cytoplasmic tails a short motif of amino acids that provides an internalization signal. Recognition of this signal by soluble cytoplasmic proteins enables the receptor and its associated ligand to be incorporated into a vesicle. Different receptors allow different rates of endocytosis of extracellular ligands.

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Background

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One model for the flow of proteins along the secretory pathway suggested that signals and receptors analogous to those in the endocytic pathway could account for the different rates at which individual secretory proteins were known to leave the ER (1417). However, attempts to identify receptors that would mediate selective export of secretory proteins from the ER met with little success.

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An alternative concept, not widely accepted at the time, was that there was no need for selective export. At the time, it was thought that all soluble proteins in the ER lumen were secretory proteins. This would mean that soluble proteins would progress along the secretory pathway in the absence of any intervention. Membrane proteins would exit the ER in much the same way, with the retention of ER-specific membrane proteins (such as the components of the translocation machinery) being explained by their incorporation into multiprotein complexes too large to fit into vesicles.

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The first sign that things were not going to be so simple came with the cloning of a cDNA encoding protein disulfide isomerase (PDI) (1413). This enzyme had been clearly shown to be resident in the ER, and to behave as a soluble protein when released from it by homogenization in vitro. In agreement with these observations, its cDNA sequence did not identify any transmembrane domain. The confirmation of PDI as a soluble ER resident protein made it necessary to refine the hypothesis that soluble proteins were nonselectively exported from the ER. Rather than propose that PDI localization required a special retention mechanism, the authors of this paper suggested that PDI might be held in the ER through a loose association with some ER membrane component in vivo.

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The experiment: KDEL is necessary and sufficient for ER localization

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Our discovery of an ER-localized protein related to the hsp70 heat shock protein focused our minds on the question of how soluble ER resident proteins were kept in the ER. This hsp70-related protein turned out to be BiP, a soluble protein known to bind incompletely assembled immunoglobulin heavy chains in the ER. Upon assembly of a complex of immunoglobulin heavy and light chains in the ER, the complex is secreted, whereas BiP remains in the ER. There followed an obvious question: how are soluble secretory proteins and ER-resident proteins distinguished from one another? We searched for clues by comparing the sequences of BiP and PDI, and noting their common features: an acidic N-terminus and the tetrapeptide KDEL (Lys-Asp-Glu-Leu) at the extreme C-terminus (1139). Sean Munro then made a suggestion that focussed our attention on the KDEL sequence alone. He had shown that BiP was identical to GRP78, one of two glucose-regulated proteins (so called because their synthesis is induced when glucose is removed and re-added to cells), the other being GRP94. Since BiP is an ER-localized homologue of one major cytoplasmic heat shock protein, he guessed that GRP94 might be an ER version of the other one, hsp90. A partial clone of GRP94 had been reported by Amy Lee some years previously, but not sequenced. We obtained it from her, sequenced it and found homology to hsp90 and, once again, the magic KDEL at the C-terminus. This could hardly be a coincidence.

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Meanwhile, we realized with some alarm that we had already done a key experiment. In order to detect BiP in vivo, we had added to it an "epitope tag," a short sequence recognized by a monoclonal antibody against human c-Myc. In the process, we had removed the last 50 or so residues of BiP, including the KDEL sequence. We had even published the result of transiently expressing this altered protein in COS tissue-culture cells — by immunofluorescence, it was in the ER (1139). This result was contrary to what was expected if KDEL were required for ER localization. Hastily, Sean repeated this experiment and found by immunoblotting that some of the myc-tagged BiP was in the culture supernatant. Radiolabelling of COS cells confirmed that the labelled BiP was secreted, although more slowly than most other secretory proteins. This result suggested that the C-terminal 50 residues might be important for the ER localization of BiP, but it also seemed possible that by truncating the protein and adding the epitope tag, we had simply perturbed the structure of BiP, and that the KDEL sequence itself was not important.

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To show that the KDEL sequence really was a retention signal, we needed to show that it was sufficient to mediate the retention of a protein that is normally efficiently secreted. For this crucial experiment we chose chick lysozyme, and engineered a series of constructs that encoded lysozyme with the c-Myc epitope tag at its C terminus. This allowed us to identify the protein by reaction with the epitope. We prepared three variants of the protein: terminating in the c-Myc epitope, or with an additional C-terminal SEKDEL, or with the control sequence SEKDAS. These constructs were expressed in COS cells.

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Figure 2  
COS cells were transfected with the cDNAs encoding lysozyme derivatives. To detect secreted proteins, culture medium from [35S]Met-labelled cells was collected, and the proteins were separated by SDS-PAGE and detected by fluorography The common band in all tracks is a protein secreted naturally by COS cells. The lysozyme derivatives differ in mobility due to the differences in the length of their C-termini. Lysozyme derivatives in cells (c) and medium (m) were detected by immunoblot analysis using the monoclonal antibody to Myc. For each experiment, equivalent amounts of cells (or medium) were analyzed.

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Figure 3  
Stained COS cells viewed with the confocal scanning laser microscope. Cells were transfected with the indicated constructs. All samples were stained with the monoclonal antibody to Myc and microscope was focussed about midway through the nucleus. ER and Golgi staining are visible as indicated.

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Figure 2 shows that by either pulse labelling or immunoblotting experiments, it was clear that addition of SEKDEL, but not SEKDAS, prevented secretion of lysozyme. Figure 3 shows immunofluorescence assays demonstrating directly that lysozyme containing the KDEL signal was in the ER. In contrast, the versions of lysozyme containing SEKDAS or only the Myc tag were concentrated in the Golgi apparatus. This Golgi localization was similar to that of wild-type lysozyme; it appeared that passage through the Golgi apparatus was a rate-limiting step for the secretion of lysozyme. As we had shown previously, BiP itself behaved quite similarly, except that it left the ER only slowly and thus was visible there even when the KDEL signal was removed (Figure 3). Experiments with other lysozyme derivatives confirmed that the KDEL signal had to be at the extreme C terminus to function, SEKDELGL being nonfunctional. A variety of other peptide sequences had no effect on lysozyme secretion, attesting to the specificity of the KDEL effect. So clear-cut were the results that we rapidly wrote them up for publication (787).

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Besides demonstrating the existence of the KDEL signal,two other features of these experiments are noteworthy. First, our studies of BiP and lysozyme were the first to use the c-Myc epitope tag recognized by the 9E10 monoclonal antibody. I had mapped the epitope for this antibody (obtained from Gerard Evan, whose lab was a few steps from mine) specifically for such use, since the first tag we developed had shortcomings (1419). Thanks in great part to Gerard's generosity in sending out the antibody, this has become one of the most widely used tags today. Second, Figure 3 is, as far as I know, the first published result obtained using a laser-scanning confocal microscope. This had been developed by John White and Brad Amos at the MRC Laboratory of Molecular Biology and, at the time we used it, consisted of a pile of equipment in a sort of tent in the workshop — the laser beam zigzagged around this structure and images were stored by simply photographing a monitor, perched precariously on top of a pile of electronic equipment, with a 35mm camera on a tripod. The spectacular power of this instrument was evident from the very beginning. By illuminating and recording the fluorescence from only a small spot at any one time, it largely eliminated the glare from out-of-focus material, allowing a 1-micron thick optical section to be clearly visualized. This revealed the presence or absence of Golgi staining with minimal interference from the surrounding ER, something that was quite impossible to do with conventional fluorescence microscopy.

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The KDEL receptor recycles

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What was the significance of the KDEL signal? At first, it argued strongly in favor of the concept of nonselective export from the ER, which was beginning to be actively promoted (281). The argument was that if a signal is required for retention even of a resident ER protein, then export is likely to occur by default, without the need for signals. Many people were surprised at this conclusion, but the logic was hard to fault. However, even in the first paper, it was evident that BiP without a KDEL signal is secreted about as slowly as the slowest secretory proteins (787). In other words, it seemed likely that other soluble secretory proteins were preferentially selected for incorporation into vesicles and secreted faster than BiP. The basis for this difference in rates of secretion remains unclear even now — whether BiP and other ER resident proteins are part of a lumenal matrix, the components of which are packaged poorly even in the absence of retrieval signals, or whether there really are export signals and receptors for all secretory proteins, is a mystery. Some good candidates for receptors have been identified, including ERGIC-53 and members of the p24 family, but they seem to be involved in the export of only a few proteins (1414; 1421). Conversely, recent studies have shown that pancreatic secretory proteins do seem to be packaged without initial concentration (1418), as predicted for a nonselective flow. However, the massive abundance of these proteins may make them a special case.

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Figure 4  
The ER contains a mixture of secretory proteins (purple) and resident KDEL-containing proteins (red). There might be some confusion about whether COPII coats act like COPI coats in selecting transported proteins via a signal. These can both leave in COPII-coated vesicles and reach the ER-Golgi intermediate compartment (ERGIC) and cis Golgi. In these post-ER compartments KDEL is recognized by its receptor (green), binding perhaps being promoted by a reduced pH, and the receptor molecules together with the KDEL-containing proteins are selectively incorporated into COPI-coated vesicles for return to the ER. Secretory proteins are not retrieved, but move on through the Golgi complex.

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A deeper impact followed from studies of the nature of the KDEL retention mechanism. To preserve the idea of unidirectional flow from ER to Golgi that follows from the concept of nonselective export from the ER, the simplest hypothesis would be that the KDEL signal acted as an anchor, binding to some immobile membrane protein in the ER. However, from the beginning we argued that the abundance of KDEL-containing proteins made this unlikely because there was no known ER membrane protein of sufficient abundance to tether all KDEL-containing proteins. We suggested instead that these proteins might escape from the ER and undergo receptor-mediated retrieval from the Golgi (787) (Figure 4). The subsequent demonstration that KDEL-tagged proteins could acquire Golgi-specific carbohydrate modifications supported this view (1420; 1412). Clearly, there was an urgent need to identify the KDEL receptor and for this we turned to yeast genetics. It turned out that most S. cerevisiae ER proteins use HDEL rather than KDEL as an ER localization signal, and by appending HDEL to the enzyme invertase, which is normally secreted, we were able to set up a screen for mutants defective for retention. These mutants secreted the HDEL-tagged invertase, which could be detected in individual colonies by a colorimetric enzyme assay. After three years hard work (and much learning by trial and error), we identified the receptor as the 7-transmembrane domain protein Erd2p (791; 790).

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The key experiment showing that Erd2p is the HDEL receptor involved a specificity swap. We found that BiP from the yeast K. lactis has DDEL instead of HDEL and that DDEL did not work well as an ER localization signal in S. cerevisiae. However, substituting the K. lactis Erd2p gene for the S. cerevisiae one allowed efficient ER localization of proteins containing a C-terminal DDEL sequence (790). Later, we found the human homologue of the Erd2p receptor, and demonstrated binding of KDEL (and HDEL) peptides to it (1415; 1425). Binding was optimal at acid pH, which suggests that pH differences between the Golgi and ER might control the association and dissociation of the KDEL ligands.

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As we had predicted, the Erd2p receptor dwelt in the Golgi and ER-Golgi intermediate compartment but recycled to the ER when bound to HDEL-containing ligand (1416). The suggestion of continuous retrograde traffic to the ER from the Golgi initially raised some objections, because it seemed incompatible with the model of nonselective anterograde transport. Nevertheless, its existence soon became accepted. More recent studies have shown that the Erd2p receptor travels in COPI coated vesicles back to the ER (see Figure 4). The Erd2p receptor binds, in a ligand-dependent fashion, to a GTPase activating protein for Arf-1 (a protein that recruits the COPI coat during vesicle formation) and this binding may regulate the selective incorporation of Erd2p into these vesicles (1411).

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The legacy

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The existence of a major, selective, retrograde transport pathway allowed new insights into the mechanisms of cargo concentration and membrane retrieval at the ER-Golgi interface. Thus, the anterograde flow of vesicular membrane carriers from ER to Golgi is largely balanced by recycling membrane via vesicles from the Golgi to the ER. This results not only in the recovery of escaped ER proteins but also, at least in some cases, in the concentration of soluble secretory cargo in the Golgi by its exclusion from the recycling vesicles (1418). Such a mechanism allows efficient delivery of cargo molecules without the need for specific forward transport signals. It is analogous to the process bywhich soluble components are thought to be concentrated following endocytosis — much of the membrane that is taken up is recycled to the cell surface whilst fluid phase components are retained in endosomes.

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More generally, the realization that the localization of soluble resident ER proteins was not a static phenomenon but rather a dynamic steady-state, in which these proteins escape to the Golgi but are efficiently retrieved to the ER, had profound consequences, reinforced by emerging findings that this was just the tip of the iceberg. Gradually, it became apparent that membrane proteins too were constantly in motion. Indeed, some of them carry KDEL/HDEL signals, though others use cytoplasmic lysine or arginine motifs that interact with the COPI coat (1422). The 1980s view of the secretory pathway as a series of fixed compartments connected by vesicles carrying cargo began to change into a much more fluid picture, in which no components stay put and organelle identity can be changed by the loss and gain of proteins. In yeast, for example, even membrane proteins, such as the SNARE Sed5p that catalyses fusion of ER-derived vesicles with the cis Golgi, are not fixed components of the Golgi but recycle through the ER. This observation suggests that cis Golgi membranes might be able to lose their identity and turn into a later Golgi compartment. Such observations paved the way for the reemergence of cisternal maturation models for transport through the Golgi stack (1423) (and see Cisternal progression occurs more slowly than vesicle movement), in which transport occurs by conversion of early cisternae into later ones rather than by vesicular transport of cargo between fixed cisternae. Though the precise contributions of vesicles are still debated, the fluidity of the system is now generally acknowledged. One can thus regard the secretory and endocytic pathways as a dynamic self-organizing system in which protein sorting determines the composition of individual membranes, and these differences in composition in turn drive the segregation and budding events which are required for sorting.

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The KDEL sequence remains one of the most blatant examples of a sorting signal known. Its conservation both within and between species is remarkable — yeast (S. cerevisiae ) contains 11 proteins with HDEL and one with KDEL; of these, three are ER membrane proteins and the rest are lumenal proteins thought to be involved in protein folding. XDEL signals (X can be a variety of amino acids, though H or K are most common) have been found in every eucaryotic species whose ER proteins have been examined. In such circumstances, to identify the second lumenal ER protein (BiP) was our good fortune. Mere comparison of the PDI and BiP sequences showed us the conserved signal. As recent entrants to the secretion field, our ignorance of the prevailing emphasis on export signals rather than retention signals also helped — we only thought to look for retention signals. But the instant success of the experiments was truly memorable. It just seemed too good to be true.

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The author

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Hugh Pelham was an undergraduate at Cambridge University, and received his Ph.D. training there with Richard Jackson and Tim Hunt. He spent two years working with Don Brown at the Carnegie Institution of Washington's Department of Embryology in Baltimore before returning to the MRC Laboratory of Molecular Biology in Cambridge in 1981. He worked initially on the mechanism of transcriptional regulation of heat shock genes, before turning to the function of the heat shock proteins. This led to the discovery of chaperones in the ER, and to studies of protein sorting and membrane trafficking throughout the secretory and endocytic pathways. Most of his recent studies have used yeast as a model organism, combining genetics with biochemistry and microscopy.


Hugh Pelham
MRC Laboratory of Molecular Biology
Hills Road
Cambridge CB2 2QH
U.K.
Phone: (44) 1223 248011
E-mail: hp@mrc-lmb.cam.ac.uk

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Last Revised on October 20, 2004<

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reviews
  • 281 Pfeffer, S. R., and Rothman, J. E. (1987).  Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi.  Annu. Rev. Biochem. 56, 829-852.  PubMed   Journal
  • 1414 Hauri, H. P., Kappeler, F., Andersson, H., and Appenzeller, C. (2000).  ERGIC-53 and traffic in the secretory pathway.  J. Cell Sci. 113 ( Pt 4), 587-596.  PubMed   Journal
  • 1422 Teasdale, R. D. and Jackson, M. R. (1996).  Signal-mediated sorting of membrane proteins between the endoplasmic reticulum and the Golgi apparatus.  Annu. Rev. Cell Dev. Biol. 12, 27-54.  PubMed   Journal
  • 1423 Pelham, H. R. (1998).  Getting through the Golgi complex.  Trends Cell Biol. 8, 45-49.  PubMed   Journal
reviews
  • 787 Munro, S., and Pelham, H. R. (1987).  A C-terminal signal prevents secretion of luminal ER proteins.  Cell 48, 899-907.  PubMed   Journal
  • 790 Lewis, M. J., Sweet, D. J., and Pelham, H. R. B. (1990).  The ERD2 gene determines the specificity of the luminal ER protein retention system.  Cell 61, 1359-1363.  PubMed   Journal
  • 791 Semenza, J. C., Hardwick, K. G., Dean, N., and Pelham, H. R. B. (1990).  ERD2, a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway.  Cell 61, 1349-1357.  PubMed   Journal
  • 1139 Munro, S. and Pelham, H. R. (1986).  An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein.  Cell 46, 291-300.  PubMed   Journal
  • 1411 Aoe, T., Lee, A. J., van Donselaar, E., Peters, P. J., and Hsu, V. W. (1998).  Modulation of intracellular transport by transported proteins: insight from regulation of COPI-mediated transport.  Proc. Natl. Acad. Sci. USA 95, 1624-1629.  PubMed   Journal
  • 1412 Dean, N. and Pelham, H. R. (1990).  Recycling of proteins from the Golgi compartment to the ER in yeast.  J. Cell Biol. 111, 369-377.  PubMed  
  • 1413 Edman, J. C., Ellis, L., Blacher, R. W., Roth, R. A., and Rutter, W. J. (1985).  Sequence of protein disulphide isomerase and implications of its relationship to thioredoxin.  Nature 317, 267-270.  PubMed  
  • 1415 Lewis, M. J. and Pelham, H. R. (1990).  A human homologue of the yeast HDEL receptor.  Nature 348, 162-163.  PubMed   Journal
  • 1416 Lewis, M. J. and Pelham, H. R. (1992).  Ligand-induced redistribution of a human KDEL receptor from the Golgi complex to the endoplasmic reticulum.  Cell 68, 353-364.  PubMed   Journal
  • 1417 Lodish, H. F., Kong, N., Snider, M., and Strous, G. J. (1983).  Hepatoma secretory proteins migrate from rough endoplasmic reticulum to Golgi at characteristic rates.  Nature 304, 80-83.  PubMed  
  • 1418 Martínez-Menárguez, J. A., Geuze, H. J., Slot, J. W., and Klumperman, J. (1999).  Vesicular tubular clusters between the ER and Golgi mediate concentration of soluble secretory proteins by exclusion from COPI-coated vesicles.  Cell 98, 81-90.  PubMed   Journal
  • 1419 Munro, S. and Pelham, H. R. (1984).  Use of peptide tagging to detect proteins expressed from cloned genes: deletion mapping functional domains of Drosophila hsp 70.  EMBO J. 3, 3087-3093.  PubMed  
  • 1420 Pelham, H. R. (1988).  Evidence that luminal ER proteins are sorted from secreted proteins in a post-ER compartment.  EMBO J. 7, 913-918.  PubMed  
  • 1421 Springer, S., Chen, E., Duden, R., Marzioch, M., Rowley, A., Hamamoto, S., Merchant, S., and Schekman, R. (2000).  The p24 proteins are not essential for vesicular transport in S. cerevisiae.  Proc. Natl. Acad. Sci. USA 97, 4034-4039.  PubMed   Journal
  • 1425 Wilson, D. W., Lewis, M. J., and Pelham, H. R. (1993).  pH-dependent binding of KDEL to its receptor in vitro.  J. Biol. Chem. 268, 7465-7468.  PubMed   Journal

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