Cell, Vol. 66, 353-364,

January

24, 1992, Copyright

0 1992 by Cell Press

Ligand-Induced Redistribution of a Human KDEL Receptor from the Golgi Complex to the Endoplasmic Reticulum Michael J. Lewis and Hugh Ft. B. Pelham MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 2QH England

Summary Resident Iuminal endqbmll reticulum (EB) proteins carry a tatgetlng stgnal (usually KDEL in animal cetls) thatatlowsthetrn$Wevatfromlaterstagesofthesecretory pathway. In yeast, the receptor that promotes this selectlve retrogcade tmnqxxt has been identtfled as the product of the ERD2 gene. We describe here the properties of a human homolog of thll protein (hEBD2). Overproduction of hEFtD2 improves retention of a protein wlth a weakly recognized variant signal (DDEL). Moreover, overexpression of KDEL or DDEL ltgands causes a redistribution of hERD2 from the Golgi apparatus to the ER. Mutation of hERD2 alters the ligand specificity of this effect, implying that it interacts directly with the retained proteins. Ligand control of receptor movement may limit retrograde flow and thus minimize fruitless recycling of secretory proteins. Introduction Maintenance of the complex structural organization of a eukaryotic cell requires that each newly synthesized protein be delivered to its correct location. For proteins that are located in the organelles of the secretory pathway, a second problem exists: they need to avoid mislocalization caused by the continual vesicular transport of membrane and luminal contents along the pathway and thus must carry signals that allow them to be retained in or returned to their place of residence. A good example is provided by the soluble proteins that are found in the lumen of the endoplasmic reticulum (ER). These carry a C-terminal tetrapeptide signal (KDEL or a related sequence) that is both necessary and sufficient for their retention in the ER (Munro and Pelham, 1987; reviewed by Pelham, 1989, 1990). This signal does not prevent their diffusion within the ER or their export from it, but allows escaped proteins to be retrieved from later compartments of the secretory pathway. That such retrieval can occur is demonstrated by the finding that proteins with an ER retention signal can undergo carbohydrate modifications that occur only in post-ER compartments (Pelham, 1988; Dean and Pelham, 1990). Genetic studies in yeast have identified the sorting receptor for luminal ER proteins as the product of the ERDP gene. This conclusion is based on three observations: mutations in erd2 cause proteins with an ER retention signal to be secreted; overexpression of ERD2 increases the capacity of yeast cells to retain such proteins in the ER; and

most significantly, replacement of the Saccharomyces cerevisiae ERD2 gene with the homolog from another yeast (Kluyveromyces lactis) alters the ligand specificity of the retention system (Semenza et al., 1990; Lewis et al., 1990). The receptor is presumed to bind its ligands in (or near) the Golgi apparatus, carry them to the ER, and then release them. Luminal ER proteins and their receptor are not the only proteins to cycle between ER and Golgi. For example, two proteins (p58 and ~58) that are normally concentrated in an intermediate compartment between ER and Golgi can be induced to move into the Golgi stack and then to the ER by appropriate temperature manipulations (LippincoWSchwartz et al., 1990; J. Saraste, personal communication). In general, however, the secretory pathway is considered unidirectional, which requires that retrograde transport from the Golgi to the ER be highly selective. The volume of reverse traffic must also be tightly regulated, both to prevent fruitless reflux of secretory proteins and to maintain Golgi structure; the very existence of the Golgi complex requires a delicate balance between membrane input from the ER, retrograde transport, and onward vesicular traffic. The possible consequences of uncontrolled membrane flow are vividly illustrated by the effects of the drug brefeldin A, which allows fusion of Golgi cisternae and the ER, resulting in a complete loss of Golgi structure and function (Fujiwara et al., 1988, 1989; Doms et al., 1989; Lippincott- Schwartz et al., 1989, 1990). Although the physiological relevance of this response remains unclear, it has been interpreted as the end result of uncontrolled retrograde transport (Lippincott-Schwartz et al., 1989, 1990). There is evidence to suggest that the ER protein sorting receptor is an important regulator of reverse transport. In yeast, retention of luminal ER proteins seems to be a dispensible function, but deletion of the ERDP gene from wild-type cells prevents their growth. Cells depleted of the ERD2 protein accumulate both intracellular membranes and the Golgi precursor of the vacuolar enzyme carboxypeptidase Y (Semenza et al., 1990). A simple interpretation of this phenotype is that cells lacking ERD2 are unable to recycle membrane efficiently and consequently fail to maintain the correct size and composition of the Golgi apparatus. In this paper, we describe studies of a human homolog of the ERDP protein (hERD2), which is 50% identical in sequence to the yeast protein (Lewis and Pelham, 1990). The results provide strong evidence that this protein is a functional sorting receptor; overexpression of hERD2 in COS cells allows retention in the ER of a protein bearing the sequence DDEL, a variant sorting signal that is normally poorly recognized in these cells. Furthermore, coexpression of a protein tagged with either KDEL or DDEL causes hERD2 to accumulate in the ER rather than in its normal location in the Golgi apparatus; we provide evidence that this response involves the direct interaction of hERD2 with its ligands. These observations suggest a

hEFtD2

P58 Figure

1. Expression

of hERD2

in Mouse

Cells

Stably transformed cells expressing c-myc-tagged hERD2 were prepared for double-label immunofluorescence compartment marker) and the anti-myc monoclonal antibody 9ElO. Note that hERD2 has the tight perinuclear complex, whereas p58 is more dispersed. The bar corresponds to 25 Km.

model in which retrograde transport of the receptor and perhaps of other membrane components is regulated by the availability of KDEL ligands. Results Localization of Expressed hERD2 To allow detection of the hERD2 protein, we expressed a version of it that was modified to include an epitope from the c-myc protein at its C-terminus. This epitope is unlikely to alter the properties of the protein significantly, since analogous tagging of the yeast ERD2 protein does not affect its function in vivo (Semenza et al., 1990). When expressed in COS cells, the human protein was found to be concentrated mainly in the Golgi apparatus (Lewis and Pelham, 1990). To see whether its distribution was affected by the high level of expression achieved in COS cells, we prepared stably transformed cell lines expressing tagged hERD2 at more physiological levels. Analysis of the hERD2 mRNA levels in these mouse cell lines showed that they were similar to those of the endogenous mFtNA in HeLacells(data notshown). lmmunofluorescenceagain revealed staining primarily of the Golgi region, together with some faint staining throughout the cytoplasm that

using rabbit anti-p58 (an intermediate distribution characteristic of the Golgi

may correspond to ER (Figure 1, ). Because it has been speculated that retrieval of KDEL proteins occurs from the so-called intermediate compartment between ER and Golgi, we performed double-label experiments with an antibody against ~58, an intermediate compartment marker in rodent cells. The pattern of ~58 staining was quite variable, sometimes being concentrated very close to the Golgi apparatus but in other experiments showing a more peripheral distribution. In the latter case it was clear that hERD2 and ~58 did not precisely colocalize; hERD2 was much more tightly confined to the presumptive Golgi region (Figure 1). Thus, under normal steady state conditions hERD2 in the mouse cell lines seems to be preferentially concentrated not in the intermediate compartment but rather in the Golgi complex. Since these results were similar to those obtained in COS cells, we conclude that the level of expression obtained from our COS cell vector does not lead to substantial mislocalization of the protein. For convenience, we used COS cells for all subsequent experiments. hERD2 Overexpression Affects ER Retention Although hERD2 isclearly homologous to the yeast sorting receptor, this alone does not establish that it performs an

Human 355

KDEL Receptor

hERD2:

KDEL --+

lysozyme

HDEL +

staining of the cells with an anti-lysozyme antibody revealed that in the absence of hERD2 the DDEL protein, like the HDEL version, was found primarily in the Golgi apparatus (Figure 3). This represents material in transit, since the Golgi staining was abolished by treatment of the cells with cycloheximide for 2 hr (data not shown). In some cells there was also a punctate staining pattern that reflects the transport of some of the lysozyme to lysosomes. Similar staining is seen when wild-type lysozyme is expressed in COS cells (S. Munro, personal communication). In contrast to this pattern, lysozyme-DDEL that was coexpressed with hERD2 was found in the ER, its distribution being identical to that of the KDEL version (Figure 3). These results are consistent with the idea that hERD2 encodes a functional sorting receptor that, when overexpressed, can recognize lysozyme-DDEL and promote its retention in the ER. However, they do not rule out the possibility that hERD2 acts indirectly, by inducing the synthesis or improving the functional efficiency of an endogeous receptor.

DDEL +

hERD2

Figure 2. Effect of hERD2 on the Cellular Accumulation Derivatives Carrying Potential ER Sorting Signals

of Lysozyme

An immunoblot containing equivalent amounts of protein from COS cells transfected with various plasmids was probed with anti-lysozyme antibodies. The plasmids expressed lysozyme with the indicated C-terminal sequences, with or without hERD2. Note that lysozyme-HDEL (arrowed) migrates somewhat more slowly on SDS- containing gels than the other constructs (Pelham et al., 1999). The basic structures of the plasmids are indicated schematically; thin lines indicate procaryotic vector sequences that are not shown in full.

analogous role in animal cells. In an attempt to demonstrate a function for the protein, we studied the effects of its overexpression on the ER retention of various reporter protein constructs. We have previously shown that COS cells can efficiently retain a KDEL-tagged version of chicken lysozyme, even when it is expressed at very high levels. However, we reasoned that proteins with an altered retention signal might be recognized less efficiently, and that their retention might be improved by increasing the level of receptor. We therefore transfected COS cells with plasmids that expressed lysozyme derivatives with the C-terminal sequences SEKDEL, FEHDEL, and YFDDEL, or with plasmids expressing hERD2 in addition to these proteins. Retention of the lysozyme derivatives was initially tested by immunoblotting of the transfected cells(Figure 2). As expected, lysozyme-KDEL accumulated to high levels in transfected cells, whereas the HDEL version did not (Munro and Pelham, 1987; Pelham et al., 1988); coexpression of hERD2 did not affect the fate of these proteins. In contrast, the behavior of the DDEL construct was dramatically affected by hERD2; when expressed alone, lysozyme-DDEL was retained poorly (though slightly better than lysozyme-HDEL), whereas in the presenceof hERD2 it accumulated to a level similar to that of the KDEL protein. This result was confirmed by analysis of the intracellular distribution of the various proteins. lmmunofluorescent

Overexpression of a Ligand Alters the Intracellular Distribution of hERD2 We next investigated whether the steady-state distribution of hERD2 was affected by the simultaneous overexpression of KDEL-tagged lysozyme. Because lysozyme accumulates to high levels and exits the ER rapidly compared with endogenous resident proteins such as BiP (Munro and Pelham, 1987), it should place a considerable burden on the retention system. The distribution of the receptor could not be followed in the experiment shown in Figure 3, because the constructs used had the c-myc epitope added to both hERD2 and lysozyme. We therefore prepared new plasmids in which the c-myc sequences were deleted from the lysozyme gene, transfected them into COS cells, and stained the cells with anti-lysozyme and anti-myc antibodies. Figure 4 shows that coexpression of an unretained version of lysozyme (with AARL at its C-terminus) did not alter the Golgi location of hERD2. Similar results were obtained with lysozyme-HDEL (data not shown). In contrast, the KDEL version caused a massive redistribution of hERD2 to the ER (note staining of the nuclear envelope in Figure 4). As a measure of this effect, we scored transfected cells for the presence of hERD2 in the Golgi apparatus. In a typical experiment, more than 80% of the cells expressing lysozyme-AARL showed clear Golgi staining (most of the rest being too faint to score), whereas only 5% of those expressing lysozyme-KDEL had detectable levels of hERD2 in their Golgi apparatus (Experiment 1, Table 1). In separate experiments, the precise fraction of the cells that werescored positive for Golgi staining varied, possibly because the growth rate or secretory activity of the cells varied, but the qualitative difference between cells transfected with the lysozyme-AARLand lysozyme-KDEL constructs was consistent and striking. Lysozyme-DDEL also altered the distribution of hERD2, but the effect was less dramatic than that induced by the KDEL protein. With our standard expression plasmids, in which both the lysozyme and hERD2 are expressed from the adenovirus major late promoter, we saw a reproducible

Cdl 356

+hERD2

-hERD2

; KDEL

HDEL

’ DDEL

Figure

3. lmmunofluorescent

Staining

of Lysozyme

in Typical

Cells from the Experiment

Shown

in Figure

2

The C-terminal sequences of the lysozyme constructs are indicated; cells in the right-hand column also expressed hERD2, while those on the left did not. The lysozyme constructs that were not retained in the ER were concentrated in the Golgi and also in lysosomes. Lysosomal staining was seen with all unretained constructs, whether or not hERD2 was present, but in this figure it is prominent only in the cell expressing hERD2 and lysozyme-HDEL. The width of each panel corresponds to approximately 140 pm.

increase in the amount of hERD2 in the ER (Figure 4), although the majority of the transfected cells still showed some Golgi staining (e.g., Experiment 2, Table 1). In an attempt to enhance the redistribution of hERD2, we expressed the lysozyme constructs from a stronger promoter (the cytomegalovirus IE promoter). This increased the amount of lysozyme and also appeared to reduce the abundance of hERD2, perhaps because of competition or interference between the transcription units. Under

these conditions, the DDEL construct significantly reduced hERD2 staining in the Golgi, although within any one experiment its effect remained less dramatic than that of the corresponding KDEL construct (e.g., Experiment 3, Table 1). The redistribution of hERD2 was a specific effect, other Golgi components being unaffected. Figure 5 shows that when hERD2 moved to the ER, the staining pattern of galactosyl transferase, a trans-Golgi marker (Roth and

Human 357

KDEL Receptor

hERD2

Figure 4. Ligand-Induced Distribution

Changes

in hERD2

COS cells expressing myc-tagged hERD2 and a lysozyme derivative with the indicated terminus were double- stained with anti-myc and anti-lysozyme antibodies. The KDEL panels are a composite: the upper part shows a focal plane that passes through the nuclear envelope, which is stained by both antibodies, while the lower part is focused on the peripheral ER.

AARL

KDEL

DDEL

Berger, 1982) did not change. As a test of Golgi function, lysozyme-KDEL was coexpressed with the lysosomal enzyme cathepsin D; the lysozyme was efficiently retained, but this did not interfere significantly with the transport of cathepsin D through the Golgi apparatus or its sorting to lysosomes (Figure 5). Taken together, the results indicate that ligands recognized by the retention system (KDEL and DDEL) cause a specific redistribution of hERD2, whereas those that are not retained (HDEL and AARL) do not have this effect. Our interpretation is that the retained proteins either bind to hERD2 in the Golgi complex and induce its movement to

the ER, or perhaps slow its export.

bind weakly to hERD2 in the ER and

A Mutant hERD2 Has an Altered Response to Ligand Although we have interpreted the previous results in terms of a direct interaction between hERD2 and retained proteins, the experiments do not rule out the formal possibility that hERD2 interacts with another cellular protein that both recognizes the ligands and moves with them to the ER. To address this question, we introduced amino acid changes into hERD2 that were designed to alter its ligand specificity

Table 1. Distribution Lysozyme Derivatives

of hERD2

consequence of its association nous, receptor.

in Cells Coexpressing

Golgi Staining (% of Transfected

Cells)

Experiment (Lysozyme Promoter)

C-Terminus of Lysozyme

wt hERD2

1 (adeno)

AARL KDEL

82” 5

2 (adeno)

KDEL DDEL

22 72

81 68

3 (CMV)

AARL KDEL DDEL

66” 0 22

70’ 34 23

Mutant hERD2

’ Transfected cells were identified by their lysozyme content, and cells that did not show Golgi staining include those in which hERD2 staining was too faint to score. With the CMV promoter present, hERD2 staining was uniformly fainter and unscorable cells more numerous.

and tested the effect of these changes on the ligandinduced redistribution of the protein. In choosing residues to mutate, we were guided by results obtained with yeast ERD2. The S. cerevisiae ERD2 protein recognizes HDEL, whereas the K. lactis homolog recognizes both HDEL and DDEL, but not KDEL (Lewis et al., 1990). Analysis of chimeras created from these two yeast receptors indicates that residues between positions 51 and 57 (K. lactis numbering) are important in determining their ligand specificity in vivo (J. Semenza, unpublished data). We therefore introduced three changes into hERD2 in this region (DLFTNYI to NLFTKYT), to make it similar to the K. lactis sequence (NLFTKWT). The altered hERD2 was then coexpressed with various potential ligands and its intracellular distribution examined by immunofluorescence. In the presence of lysozyme-AARL (Figure 6) or lysozyme-HDEL (data not shown), the mutant hERD2 behaved as normal, concentrating in the Golgi apparatus. However, in contrast to the wild-type protein, its localization was almost unaffected by coexpression of lysozymeKDEL (Figure 6; Table 1). Only when the cytomegalovirus promoter was used to increase expression of lysozymeKDEL was redistribution observed in some of the cells (Experiment 3, Table 1). On the other hand, the mutations did not affect the movement of hERD2 in response to lysozyme-DDEL (Figure 6, Table 1). Furthermore, the mutant protein retained its ability to prevent secretion of DDELtagged lysozyme as judged by immunofluorescence (note the ER location of lysozyme-DDEL in Figure 6) and also by immunoblotting of transfected cell samples (data not shown). Table 1 shows that the mutations significantly altered the apparent ligand specificity of hERD2; whereas the wild-type protein responded more strongly to KDEL than to DDEL, the mutant showed little discrimination and, if anything, the opposite bias. The ligand-specific nature of this effect strongly suggests that hERD2 interacts directly with the lysozyme constructs, and that its movement is caused by this interaction rather than being an indirect

with some other, endoge-

Gradients of hERD2 within the ER When examining cells coexpressing hERD2 and lysozyme-DDEL, we observed that the distribution of hERD2 within the ER was frequently nonuniform when compared with that of the retained lysozyme (which, being soluble, should be free to fill the ER lumen evenly). This is noticeable in the bottom panel of Figure 6, and more dramatic examples are shown in Figure 7. Typically, the cells showed some Golgi staining and a gradient of hERD2 within the ER, with the highest concentration around the Golgi apparatus. Such cells were most common in those experiments in which the the ligand-induced redistribution of hERD2 was particularly dramatic. The gradients could not easily be explained by artifacts of microscopy or photography. They were clearly discernable in the digital output of a confocal laser-scanning microscope, adjusted to collect both fluorescent images simultaneously with a single excitatory laser beam (Figure 7). Controls in which cells were incubated with antilysozyme antibodies followed by a mixture of Texas Red and FITC-labeled second antibodies showed that the optical system could not generate comparable (apparent) gradients where none existed, as is also implied by the fact that gradients were not observed in all cells. Furthermore, although it was difficult to be certain that all cytoplasmic fluorescence emanated from the ER, comparison of the red/green fluorescence ratios of the nuclear envelope and of reticular ER at the cell periphery, both of which were clearly identifiable, confirmed the unevenness of the hERD2 distribution. Gradients of hERD2 were never observed when unretained versions of lysozyme were expressed. Surprisingly, they were also difficult to detect in cells expressing lysozyme-KDEL, even though these were treated identically to those shown in Figure 7. This argues against some trivial explanations of the observed gradients, such as the existence of a subset of cells whose structure or physiology somehow prevents the receptor from spreading throughoutthe ER, regardlessof its function. Asdiscussed below, the distribution of the receptor is more likely to reflect its precise itinerary within the cell, which may in turn be influenced by the available ligands. Discussion Evidence That hERD2 Is a Functional Sorting Receptor The hERD2 protein analyzed in these experiments was previously suggested to be a KDEL receptor on the basis of its homology with the yeast fffD2 gene product. We have now shown that hERD2 has two further properties that are consistent with its postulated sorting role. First, retention of a DDEL-tagged protein in the ER of COS cells, which is normally an inefficient process, is greatly improved byoverexpression of hERD2. Second, overexpression of lysozyme derivatives bearing either KDEL or DDEL

Human 359

KDEL

Receptor

hERD2

GalT

KDEL

lysozyme-KDEL

cathepsin D

Figure

5. Lack of an Effect

of Lysozyme-KDEL

on Golgi

Structure

and Function

COS cells expressing myc-tagged hERD2 and lysozyme-AARL or -KDEL (as indicated) were stained with anti-myc (left) and anti-galactosyl transferase antibodies (right). To preserve the immunoreactivity of the galactosyl transferase, the cells were fixed with methanol and acetone, which does not preserve ER structure well. The bottom panel shows a cell coexpressing myc-tagged cathepsin D and lysozyme-KDEL stained with anti-myc (left) and anti-lysozyme (right). The cathepsin D can be seen in the Golgi apparatus and in (pre)lysosomes. The same distribution is seen when tagged cathepsin D is expressed alone (Pelham, 1959).

hERD2

lysozyme

Figure 6. Distribution of Mutant pressed with Potential Ligands

AARL

hERD2

Coex-

This figure is equivalent to Figure 4, except the version of hERD2 containing 3 amino acid changes was used. Note that the distribution of this mutant protein was affected by lysozymeDDEL, but not by lysozyme-KDEL. Note also that it caused lysozyme-DDEL to be retained in the ER. The top panel has a slightly higher magnification than the other two panels; bars are 25 urn.

KDEL

DDEL

induces movement of hERD2 from the Golgi complex to the ER. The ligand specificity of this response can be altered by mutation of the hERD2 protein itself, which strongly suggests that proteins bearing a retention signal interact with hERD2 in adirect and highly specific manner. Although these results provide good evidence that hERD2 is a KDEL receptor, they do not prove that it is the only one. Indeed, we have recently identified a second human cDNA clone that encodes a protein closely related to, but distinct from the hERD2 protein studied in this work

(M. Lewis, unpublished data). In addition, a quite different putative KDEL receptor has been identified by an antiidiotypic antibody approach (Vaux et al., 1990). Further studies will be required to define the precise role of each of these proteins. Signal ‘Transduction by the hERD2 Molecule The observation that the distribution of hERD2 can be controlled by the presence of KDEL- or DDEL-containing proteins implies that the information that ligand is bound,

Rumafl 361

KDEL Receptor

lysozyme

-0

1ysozyme

l\.&J

-27-xF-zT-

“,z--8,

distance(microns) Figure 7. Gradients of hERD2 within the ER Examples of cells coexpressing hERD2 and lysozyme-DDEL (A) and (B) are shown, each double-labeled to show hERD2 (left) and lysozyme (right). by the CIonfocal Note that hERD2 was typically present both in the Golgi and in the nearby ER. (C) shows plots of the pixel intensity recorded microscope in each fluorescence channel along the line indicated in the right-hand part of (B). The black bar indicates the position n of the nl ucleus, and the circle indicates the closest approach to the Golgi apparatus (the line does not pass through the Golgi apparatus itself).

on the luminal side of the membrane, can be transmitted to the vesicular transport machinery, which is likely to be predominantly located in the cytoplasm. This signaling property must depend on the structure of the receptor, which at present can only be deduced by examination of its primary sequence. The sequences of the three known ERD2 proteins (S. cerevisiae, K. lactis, and human) share a common pattern of seven predominantly hydrophobic stretches alternating with regions rich in unconserved polar amino acids (Lewis and Pelham, 1990). This suggests a structure analogous (though probably not homologous) to that of the bacteriorhodopsin family of seven-transmembrane-domain proteins (Henderson et al., 1990). Binding of ligand could occur either to surface loops, or more likely to a pocket in the protein lined with some of the conserved polar residues that are found within the putative transmembrane domains. In either case, it is easy to imagine a ligand-induced conformational change that would affect a large part of the protein and alter its interaction with the cytoplasmic transport machinery. Receptor Movement To perform its function as a sorting receptor, hERD2 must cycle between some post-ER compartment, where it binds ligand, and the ER, where the ligand should be released. Under normal circumstances most of the receptor (presumably unoccupied by ligand) accumulates in the Golgi apparatus. Double labeling suggests that it is primarily in the Golgi complex itself, rather than in the intermediate compartment identified by the ~58 marker protein. Coexpression of an artificial ligand causes the receptor to accumulate instead in the ER, implying that ligand binding either increases its rate of movement to the ER, or slows its exit from there, or both. In principle, an increase in the KDEL concentration in the ER could increase the occupancy of receptor in this organelle; if occupied receptor is inefficiently exported relative to the unoccupied form, a net shift of receptor to the ER would occur. However, it seems unlikely that expression of an additional ligand could have such a striking effect, given the high levels of KDEL proteins already present. Based on previous estimates of protein levels achieved with the vectors used in this work (Munro and Pelham, 1987) and the assumption that BiP comprises about 20% of the luminal ER protein, we calculate that the total KDEL content could be increased at most about three-fold; if the volume of the ER lumen increased in response to this, the change in KDEL concentration would be even less. On the other hand, expression of lysozyme-KDEL would be expected to have a dramatic effect on the rate at which KDEL ligands arrive at the Golgi apparatus; without KDEL, lysozyme leaves the ER about ten times more rapidly than BiP (Munro and Pelham, 1987), and other luminal ER proteins are also slow to leave the ER when their retention signals are removed (Mazzarella et al., 1990). Thus, when lysozyme-KDEL is expressed, receptor molecules in the Golgi apparatus should be rapidly filled with ligand; if occupied receptor is selectively and efficiently incorporated

into retrograde transport vesicles, its redistribution under these conditions can readily be explained. These possibilities are not mutually exclusive, and it is perhaps easiest to imagine hERD2 existing in two states: a ligand-free form that is efficiently transported from ER to Golgi, and an occupied form that is selectively recognized by the retrograde transport machinery. Unoccupied receptor must also carry signals that prevent its movement beyond the Golgi apparatus. These may be distinct from the recycling signals; alternatively, at some point in the secretory pathway the conformational change normally induced by ligand may occur spontaneously, allowing unoccupied receptor to be retrieved. A striking feature of the receptor distribution was the apparent gradient of its concentration within the ER of some cells that coexpressed lysozyme-DDEL. Analogous gradients in the plasma membrane have been observed for several recycling cell surface receptors (Bretscher, 1983; Bretscher and Thomson, 1983), and their existence has been used as an argument that delivery and removal of the receptors is occuring at distinct sites. In the case of hERD2, a gradient could be formed if retrograde transport delivers the receptor to the ER in the vicinity of the Golgi apparatus, while at least some forward transport occurs from more peripheral sites. There is indeed microscopic evidence for peripheral ER exit sites (Schweizer et al., 1990; Chavrier et al., 1990; Duden et al., 1991; Saraste and Svensson, 1991) and the dimensions of the gradients are compatible with this model. hERD2 is typically concentrated within 20 pm of the Golgi, which may correspond to a distance of 30 or 40 pm along the ER membrane; if it has a diffusion coefficient similar to that of rhodopsin (Poo and Cone, 1974) it would take lo-15 min for hERD2 to diffuse such a distance, a time comparable to that spent in the ER by other efficiently transported membrane proteins (e.g., Puddington et al., 1986). The hERD2 gradients could thus plausibly be generated by ligand-induced recycling between Golgi and ER. However, other possibilities are not excluded by the present data. Cells expressing lysozyme-KDEL seldom exhibited a gradient of hERD2 in the ER. One possible explanation for this difference is that at least some of the receptor binds to lysozyme-KDEL soon after leaving the ER, and recycles without passing through the Golgi complex. Given that ligand binding affects receptor movement, it does not seem implausible that ligands that differ in their affinities and/or their optimal ionic conditions for binding might have different effects on the precise pathway of receptor movement. This argument leads to the prediction that different ER proteins might be retrieved from different parts of the secretory pathway, depending on the conditions required for their binding to the receptor. Interestingly, although most evidence indicates that ER proteins do not pass beyond the cis-Golgi (Pelham, 1989) in rat liver, the KDEL protein calreticulin has been reported to acquire galactose residues, implying passage to the trans-Golgi (Van et al., 1989). Thus, it may be impossible to define a unique “salvage compartment” in animal cells from which all ER proteins are recycled. Instead, ER proteins may be pro-

Human 363

KDEL

Receptor

gressively extracted from a series of compartments, an arrangement that should increase the efficiency of the sorting process (Rothman, 1981). Regulation of Retrograde Transport One further possibility raised by our results is that retrograde flow in general may be modulated by the availablility of KDEL proteins, In yeast, the ERD2 gene product appears to function not only as the receptor for luminal ER proteins but also as an essential component of the retrograde transport machinery (Semenza et al., 1990). Whether this essential function of the receptor is dependent on its interaction with ligands is not clear; viable mutants exist that fail to retain ER proteins, but it is possible that receptor trafficking is altered in these strains. Nevertheless, the dual role of ERD2 suggests a model in which cells use the efflux of resident proteins from the ER as a measure of the rate of vesicular traffic into the Golgi; increased forward flow would produce more ligands for the Golgi pool of receptor, stimulating its recycling and thus leading to an increase in the return flow. Conversely, when forward transport and hence the supply of KDEL proteins was diminished, there would be a corresponding reduction in reverse transport. In this way, the return of crucial membrane components (such as those involved in vesicular transport itself) would be assured, while reflux of secretory proteins would be kept to a minimum. Experimental

Procedures

Plasmids The basic structures of the lysozyme and hERD2 expression plasmids are indicated schematically in Figure 2. For expression of iysozyme derivatives alone (Figure 2 and Figure 3), we used piasmids HYH and HYK (Pelham et al., 1988) and a similar plasmid, HYD. These express chick lysozyme with the last two amino acids replaced by the sequence PCM, followed by the c-myc epitope EQKLISEEDL. and then NSEKDEL (HYK), NFEHDEL (HYH), or NYFDDEL (HYD). Coexpression of hERD2 was achieved by inserting an additional fragment coding for a c-myc tagged version of hERD2 from the expression plasmid described by Lewis and Peiham (1990), to form piamids HYHE, HYKE, and HYDE. The piasmids used for the receptor movement experiments (Figures 4-7) were of similar construction but lacked the c-myc epitope on the iysozyme moiety, thus allowing unambiguous detection of the tagged receptor. in the three basic piasmids used, the lysozyme moiety was followed by PNSEKDEL (HYKE4), PNSAEAARL (HYAE4) (both made by fusing Nsil and EcoRl sites flanking the c-myc epitope), and PCMYFDDEL(HYDM)(made by inserting an appropriate oligonucleotide at the C-terminus of the lysozyme coding sequence). Versions of these piasmids were also constructed with the hEAD moiety carrying the three point mutations described in the text. This was done by elongating a suitable mutagenic primer on a single-stranded hERD2 template (Kunkel et al., 1987) in a Bluescript vector and replacing the wild-type hERD2 with the mutant, generating plasmids HYKE5. HYAES, and HYDE5. The cytomegaiovirus IE promoter-driven constructs described in Table 1 were derived from the HYXE4 and HYXE.5 plasmids and have the Sacii-Hindlll fragment that contains the adenovirus promoterdriving iysozyme expression replaced with the Nrul-Hindlll CMV promoter fragment from plasmid CDMB (Seed, 1987). The cathepsin D-lysozyme expression plasmid (CDYK) used for the lower panel of Figure 5 was derived from the myc-tagged cathepsin D expression plasmid CDM (Peiham, 1988) and has an adenovirus ML promoter, untagged lysozyme-KDEL coding sequence, and herpes tk poly(A) site inserted at the Sacll site. This generates tandem iysozyme and cathepsin D coding sequences, each with the adeno promoter and

herpes tk poly(A) lysozyme-hERD2

site, in an arrangement coexpression plasmids.

analogous

to that of the

Generation of Stable Cell Lines Expressing hERD2 The plasmid used for the construction of stable lines was similar to the HYXE series, but the lysozyme expression cassette was replaced by a neomycin resistance gene cassette from pPyNeo (A. Smith and M. Goodeli, unpublished data) comprising a polyoma virus origin and enhancer, the neo coding sequence, and an SV40 poiyadenyiation site, oriented with the neo transcription unit in the opposite direction to the hERD2 transcription unit. The plasmid was linearized at the unique Sfii site in the SV40 origin and transfected into the WOP 32-4 mouse cell line (Dailey and Basilica, 1985) by electroporation. The cells were allowed to recover for 48 hr and then resistant clones were selected on 400 @/ml G418. Resistant clones (named TSE ceils) were screened for hERD2 expression by immunofluorescence using the monoclonal antibody 9ElO. The parental ceil line and the TSE lines generated express a temperature-sensitive allele of the poiyoma large T antigen, but they were maintained continuously at the nonpermissive temperature (39OC) for the experiments reported here. COS Cell Transfections and immunofiuoreecence Analysis COS cells were transfected as described previously (Munro and Pelham, 1987), transferred toslidesonedayiater, and fixed4860 hrafter transfection. Fixation of both COS and mouse cells was performed with 2% formaldehyde, 0.2% giutaraldehyde for 15 min, or sometimes with 1% giutaraidehyde. The presence of glutaraldehyde proved crucial for the preservation of ER structure, and especially for effective fixation of hERD2. The cells were permeabilized with 0.5% Triton X-100 in phosphate-buffered saline for 10 min, treated with 1 mg/ml NaBH, in phosphate-buffered saline for IO min, and then stained as described previously (Munro and Pelham, 1987). For staining with antibodies to gaiactosyi transferase, cells were fixed at -2OOC in methanol (6 min) and then acetone (30 s). Double labeling was performed using either the anti-myc monoclonai antibody 9ElO (Evan et al., 1985) and polyclonai rabbit antilysozyme, or rabbit antibodies raised against a synthetic version of the myc epitope and the D1.3 anti-iysozyme monoclonai antibody (Mariuzza et al., 1983). Rabbit antibodies against gaiactosyi transferase were the kind gift of E. Berger (Roth and Berger, 1982), and anti-p58 antibodies were generously provided by J. Saraste (Saraste et al., 1987). FITC- or Texas Red-labeled sheep anti-mouse ig and donkey anti-rabbit lg (Amersham) were used as second antibodies. Images were obtained using an MRC-500 confocal laser-scanning microscope (BioRad). FITC and Texas Red were excited with a single laser beam, and red and green images collected simultaneously using appropriate filtration. This ensured that the images came from the same focal plane and greatly reduced the potential problem of photobleaching. Because bleed-through of bright FITC signals to the Texas Red channel could occasionally be detected, whereas the converse could not, we always used Texas Red labeling for the brighter signal (usually from lysozyme). The scoring of cells for the presence of hERD2 in the Golgi (Table 1) was performed using a Zeiss Axiophot microscope. Transfected ceils were first selected using the lysozyme channel, and then scored for the presence of Goigi staining that was stronger than that of the surrounding ER. More than 100 ceils were examined for each sample, and the percentage of transfected cells that were positive by this criterion was calculated. For some cells, no hERD2 staining above background could be detected (the detection efficiency for hERD2 being apparently much lower than for lysozyme). These cells were scored as “no staining of the Golgi” rather than being ignored, because we observed that when hERD2 was distributed over the large area of the ER, it was much harder to detect than when it was concentrated in the Goigi, which means that discarding apparently negative ceils would introduce an unjustified bias in favour of Golgi staining. Acknowledgments We thank Eric Berger and Sally Ward for anti-galactosyl transferase and anti-lysozyme antibodies, respectively: Mark Bretscher for helpful

Cdl 364

discussions about protein diffusion and recycling; and Sean Munro and Jan Semenza for unpublished information and comments. The costs of publication of this article were defrayed in part by the payment of page charges. This artcle must therefore be hereby marked “advertisement” in accordance with 18 USC section 1734 solely to indicate this fact. Received

September

9, 1991; revised

October

23, 1991

contains three copies of the active site sequences isomerase. J. Biol. Chem. 265, 1094-1101.

Munro, S., and Pelham, H. R. B. (1987). A C-terminal secretion of luminal ER proteins. Cell 48, 699-907.

disulphide

signal prevents

Pelham, H. R. B. (1988). Evidence that luminal ER proteins from secreted proteins in a post-ER compartment. EMBO 918. Pelham, H. R. B. (1989). Control of protein reticulum. Annu. Rev. Cell Biol. 5, l-23.

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