Recycling

of proteins between the endoplasmic and Colgic complex

reticulum

Hugh R;B. Pelham MRC Laboratory

of Molecular

Biology,

Cambridge,

UK

Several lines of investigation have shown that protein transport from the endoplasmic reticulum to the Golgi is more complex than previously imagined. Dynamic sorting of both membrane and soluble proteins is now believed to occur on the cis side of the Golgi apparatus with some proteins returning to the endoplasmic reticulum while others travel onwards.

Current

Opinion

in Cell Biology

Introduction The vesicular transport of proteins through the cell creates a series of sorting problems because the composition of each compartment has to be maintained despite the flux of secretory and membrane proteins through it. This problem is well documented in the case of the endoplasmic reticulum (ER): viral glycoproteins can leave the ER within 10-15 min of their synthesis, while resident ER proteins maintain their location for days. The current view is that export of proteins from the ER does not depend on any speciiic signal, but occurs by a continuous bulk-flow mechanism. In contrast, luminal ER proteins are retained by a specific mechanism that depends upon the presence of a carboxy-terminal tetrapeptide signal, Lys-Arg-Glu-Leu (KDEL in the single-letter amino-acid code) or a closely related sequence. Theoretical considerations have led us to propose that KDEL is not literally a retention signal, but instead allows the receptor-mediated retrieval of the proteins from a postER compartment [ 11. This implies retrograde vesicular transport from the Golgi complex, or some Intermediate compartment, to the ER In the past year, good evidence for such retrograde transport has accumulated, and its existence is now generally accepted. In this review, I focus on the evidence for recycling and its implications. Other recent reviews cover the problems of protein transport from the ER more comprehensively [2,3].

CGtiis-Golgi

1991, 3:585-591

Recycling proteins

of luminal

Biology

reticulum

The easiest way to track the movement of proteins within the secretory pathway is to examine the carbohydrate mod&cations that they acquire, and this approach has been used to demonstrate that KDEL-containing proteins can, transiently, leave the ER Thus, when a KDELtagged version of the lysosomal enzyme, cathepsin D, was expressed in COS cells, it accumulated in the ER but was still mod&d by the enzyme, N-acetylglucosaminel phosphotransferase [ 41. As the phosphotransferase is believed to be in (or near) the &Go&i, this suggested recycling of the cathepsin through this compartment. It is, however, diI3cult to rule out the possibility that there is a small amount of phosphotransferase in the ER, and that this accounts for the observed modification. This objection has been overcome by performing an analogous experiment in yeast (Saccharomyces 133-e vtie). Fusion proteins consisting of pro-a-factor or inw-tax tagged with His-Arg-Glu-Leu @DEL), the retention signal in this organism, were shown by subcellular fractionation to be located In the ER, but they were modiiied by the addition of al-&linked mannose residues [5*]. This modilication occurred in a post-ER compartment, as could be shown by using a temperature-sensitive Secl8 strain. At the non-permissive temperature, vesicle fusion is blocked in such suains; under these conditions,

Abbreviations network; ER-endoplasmic reticulum; PDl-protein

@ Current

endoplasmic

Ltd ISSN 0955-0674

disulphide

isomerase.

586

Membranes

the fusion proteins were trapped in the ER: and failed to receive al4inked mannose residues [5*]. These experiments provide very strong evidence that HDEL-tagged proteins normally leave the ER, undergo Golgi-specific modifications, and then return. In yeast, the precise site from which retrieval occurs seems to vary depending on the growth conditions. In minimal medium, HDEL proteins exhibit only an early Golgi modiiication (addition of a few mannose residues), but in rich medium, more extensive outer-chain modification occurs, implying deeper penetration of the Golgi complex. ConIirmation of this is obtained from studies of er-dl mutants, which are defective ln outer-chain modiIication [6=]. On rich medium, such mutants also fail to retain FIDEL proteins; however, on minimal medium, retention is unimpaired, presumably because ER proteins do not reach the defective Go@ compartment.

KDEIJHDEL

receptors

Further evidence for recycling is provided by the identification and localization of the KDEI&IDEL sorting receptors in yeast and animal cells. The yeast receptor has been identified by genetic means [7*,8*]. An extensive screen yielded only one gene that was required for the retention of FIDEL proteins under all growth conditions. This gene (ERD2) encodes a 26 kD protein which is predicted to have seven transmembrane domains [9-l (it does not, however, show sequence homology to the rhodopsinlike family of cell-surface receptors). When overexpressed, the ERD2 product can increase the HDEL retention capacity of wild-type yeast cells [7*]. Moreover, when the S. cerer~isr& ERD,? gene is replaced by the homologue from the related budding yeast, Kluyueromyces kutk, the signal specificity of the retention system alters such that both DDEL and HDEL are recognized, instead of HDEL alone [ 8.1. This is consistent with the presence of both HDEL and DDEL on K kzctrj ER proteins [8*], and indicates that the ERD2 gene is responsible for the speciIic recognition of the retention signal. Together, these genetic experiments argue very strongly that ERD2 encodes the sorting receptor. A homologue of the ERLl2 gene product with 50% amino acid sequence identity to the yeast proteins has recently been isolated from human cells and, on the basis of this homology, is proposed to be the human KDEL receptor [p]. Subcellular fractionation and immunofluorescence studies of epitope-tagged versions of both the yeast and human ERD2 proteins have shown that they are present at highest concentrations, not in the ER, but in structures indistinguishable from the Golgi complex [7*,9*]. In mammalian cells, some dispersed vesicular staining has also been found, and the protein was detectable in the ER in at least some cells. This distribution is consistent with a recycling mechanism for ER protein retention; in the steady state, it would be advantageous for an unoccupied receptor to be present in the Golgi complex, which is

ready to bind escaped proteins and carry them back to the ER At present, it is unclear how the movement of the receptor and the binding and release of ER proteins are regulated. A rather different approach to identify the KDEL receptor was pursued by Vaux et al [lo*], who prepared antibodies against KDEL-containing peptides, and used these to obtain anti-idiotypic monoclonal antibodies. These antibodies recognized a 72 kD glycoprotein on western blots of mammalian cells, and the hybridoma cells themselves secreted very large amounts of a soluble form of this protein. This soluble protein bound to protein disulphide isomerase (PDI), an ER protein, and also to KDEL-containing peptides. Immunofluorescence studies indicated that the 72 kD glycoprotein was present in a post-ER compartment, most probably in an intermediate structure befween the ER and the Golgl cistemae (the c&Golgi network - see later) [lo*]. These properties are consistent with the idea that the 72kD protein is a KDEL receptor, and raise the possibility that this protein and the ERD2 gene product both play a role in the retention of ER proteins in animal cells. However, some caution is necessary. The binding activity observed for the soluble form of the 72 kD protein was very weak - the & for PDI (2&50 @I) implies a half-life for the complex of only a few milliseconds. Moreover, the hybridoma cells producing this protein did not appear to secrete ER proteins, as would be expected if it could bind KDEL sequences in vivo and mask them from the retrieval system [lo*]. Further studies of both the 72 kD protein and the human Ef7D2 gene product are in progress, and should help to define their precise contributions to the retrieval process.

A retention membrane

signal for endoplasmic proteins

reticulum

If resident luminal ER proteins can recycle through the Golgi, it is at least formally possible that ER membrane proteins do the same. So far, only one known membrane protein is believed to use the KDEI&IDEL system - this is the product of the yeast SEC20 gene; it is involved in vesicular transport from ER to Golgi and its function may require it to shuttle between these compartments. It spans the membrane once and has a carboxy-terminal HDEL sequence that is located, appropriately, on the luminal side (D Sweet, H Pelham, unpublished data) [ 111. A quite distinct retention signal was iirst identiIied on the short cytoplasmic tail of the adenovlrus E3/19K protein, which accumulates ln the ER of infected cells. This sig nal is sufficient, when transplanted, to retain other transmembrane proteins such as CD4+ and CD8+ in the ER [12]. Recent extensive mutational analysis has deIined the signal as the carboxy-terminal sequence Lys-Lys X-X (KKXX) or Lys-X-Lys-X-X (KXKXX); surrounding sequences can all be replaced by serine residues [ 13.1. The motif is present on certain other resident ER membrane

Recycling

of proteins

between

proteins, although it may not be the only retention signal on them. Such a speciIic signal suggests an equally speciiic receptor, and an obvious possibility is that the receptor would act to retrieve protein molecules that leave the ER This model is favoured by Jackson et al. [ 13.1, but so far no firm evidence for recycling of proteins bearing the KKXX signal has been published.

The effects of brefeldin

A

A widely cited argument in favour of retrograde transport from the Golgi complex to the ER comes from studies of the drug, brefeldin A, which was found some years ago to block protein secretion and cause disruption of the Golgi structure [ 141. More detailed studies have shown that soon after addition of the drug, long tubules extend from the Golgi complex, in a microtubule-dependent manner, and fuse with the ER [ 15.1. As a consequence, many of the enzymes normally present in the Golgi are delivered to the ER [ 15*-191. It has been suggested that retrograde transport normally occurs via this pathway, and that the effects of brefeldin are due to the inhibition of forward transport without a corresponding inhibition of retrograde flow, resulting in excessive movement of Golgi proteins into the ER [ 150,161. Subsequent work showed that brefeldin A causes the rapid dissociation of a protein termed P-COP from Golgi membranes [20-l. This protein is a component of the coat structure found on Golgi cistemae and associated transport vesicles; it shows weak amino acid sequence homology to the P-adapt-ins, which are components of clathrin-coated vesicles [ 21*,22*]. Coat proteins could be required for the pinching-off of a budding tubule to form a discrete vesicle, and/or to prevent the fusion of budding vesicles with their targets before budding is complete. If so, it is easy to see how brefeldin A could allow long tubules to form and fuse with other compartments, thus joining Golgi cistemae with each other and with the ER Such connections would not be entirely random, be ing dependent on the molecules that normally determine the specificity of membrane-fusion events. It seems unlikely that retrograde transport normally involves continuous tubular connections between Golgi and ER However, the tubules that emanate from the ch Golgi in the presence of brefeldin A could represent vesicles travelling to the ER which have failed to pinch off and thus have been drawn into the tubules. Unfortunately, other interpretations are also possible: the tubules could be formed by a process that spreads the c&Go@ into a dispersed network (see later), or could result from an abnormal association of microtubule motors with Golgl membranes in the absence of coat proteins. The subsequent tubular connections to the ER could reflect fusion events that are specific to retrograde vesicles, or merely those that must occur during forward transport. Thus, although brefeldin A provides important clues about the operation of the secretory pathway, it seems dangerous

the endoplasmic

reticulum

and Golgi

complex

Pelham

to assume that it reveals the normal retrograde flow from Golgi to ER

Recycling

and the cis-Golgi

network

There is currently much interest in the idea that a distinct compartment exists between the ER and the Golgi stack in animal cells, and that this structure, variously known as the c&Golgi network, the intermediate, 15’C, or salvage compartment, is the site from which ER components are retrieved [ 31. In this review, I shall refer to the structure as the &-Go@ network (CGN), a term that implies no particular function; however, I do not mean to imply that the various elements of the structure necessarily form a connected network at any one time. The concept that the CGN has a distinct identity rests largely on the 6nding that two membrane proteins (~58 [ 231 and p53 [ 241) seem normally to be resident in this structure, and they are increasingly used to define it. Immunoelectron microscopy shows that these proteins are concentrated in tubules and vesicles close to the cb side of the Golgi, although they are also observed within the first Golgi cistema and in membranous structures that have a peripheral location, often far from the Golgi apparatus [23,24]. Occasionally, they are also seen in parts of the rough ER Immunofluorescence studies in tissue culture cells have revealed considerable variations in the distribution of the CGN proteins under different conditions. Incubation at 15°C causes both p53 and p58 to become much more closely associated with the Golgi complex, and the peripheral structures also become more prominent (T Saraste, personal communication) [ 15*,25*]. In cells that are infected with vesicular stomatitis virus or Semliki Forest virus and which are then Incubated at low temperature, the peripheral elements co-localize with viral glycoproteins arrested in transit to the Golgi (T Saraste, personal communication) [ 250,261, which in turn co-localize with P-COP [ 21.1. These structures are therefore likely to be associated with sites at which transport vesicles bud from the ER. Upon return from 15’C to 37”C, both p53 and p58 can briefly be detected in the ER before they revert to their normal distribution (T Saraste, personal communication) [ 15.1. Such observations strongly suggest that these proteins are able to cycle between Golgi and ER The peripheral elements of the CGN may simply be the outposts of a dispersed but functionally homogeneous network, but it is also possible that they are, or are derived from, the actual transport vesicles that carry proteins from the ER Their membrane components could bud from the ER, perhaps fuse to form larger structures, travel to the c&Golgi region, and fuse with the Golgiassociated portion of the CGN, and then return to the ER in retrograde transport vesicles. The first part of this pathway would be identical to that followed by plasma membrane and secretory proteins, but instead of progressing

587

588

Membranes

over, rapidly transported proteins become concentrated as they proceed - Semliki Forest virus glycoproteins, for example, are some eightfold more concentrated in the Golgi than in the ER [27]. In ceils incubated at low temperature, concentration of viral glycoproteins is apparent even in the CGN, including its peripheral elements [21*,25*,26]. If forward transport occurs entirely by bulk Bow - that is, transported proteins are free to diffuse into and out of transport vesicles as they form and fuse then the existence of retrograde vesicular transport poses a problem: how do proteins such as vesicular stomatitis virus glycoprotein avoid returning to the ER?

Fig. 1. Apparent cis-Go@ network,

relationships and the

of the endoplasmic Colgi stack.

through the Golgi stack, CGN components tinually recycle through the ER (Fig. 1).

reticulum,

the

would con-

The KDEL receptor(s) would be expected to follow essentially the same route, but their steady-state distribution need not be the same as that of p53 and ~58. In deed, the human ERD2 homologue (presumptive KDEL receptor) seems to be much more closely associated with the Golgi complex than with the CGN (M Lewis and H Pelham, unpublished data) [9*]. Whether retrieval of ER proteins actually occurs from the CGN is difficult to say, given the scattered and variable nature of this ‘compartment’. It seems reasonable, however, to assume that luminal ER proteins and CGN components recycle by the same pathway, and thus at some point are present in the same structures.

Maintenance

of forward

transport.

For many proteins, transport along the secretory path way appears to be both rapid and unidirectional. More-

For a strict bulk-flow model, this problem is a severe one. Concentration of proteins in the CGN requires the rate of non-selective vesicular transport of proteins into this compartment to be substantially greater than the rate at which they exit, which means that more vesicles must fuse with the CGN than bud from it. This leaves a large amount of material, particularly Lipid, that somehow has to be returned to the ER without secretory proteins travelling with it. Wieland et al. [28] proposed that this lipid is recycled one molecule at a time, by a mechanism involving phospholipid transfer proteins. Intriguingly, the yeast SEC14gene, which is normaLly required for protein transport through the Golgi complex, was found to encode a phosphatidylinositol-phosphatidylcholine transfer protein, lending some support to this hypothesis [29’]. Mutations in several other genes, however, notably the gene that encodes choline kinase, can restore normal secretion to cells that completely lack the SEC24 gene [300]. Because the double-mutant cells have no detectable transfer activity for phosphatidylcholine or phosphatidylinositol (the major yeast phospholipids), such an activity is unlikely to be required for the secretory process. In order to explain transport entirely in terms of bulk vesicular flow, it is therefore necessary to postulate an entirely novel Lipid retrieval mechanism of high capacity (Fig. 2). The only obvious alternative would be for rapidly transported proteins to be bound by some kind of receptor(s) [31]. These receptors do not necessarily have to show great specificity or a very high affinity; they could either bind (and thus concentrate> proteins in the ER and move with them into transport vesicles, or hold them in the CGN while excess membrane is removed in vesicular form (Fig. 2b and 2~). At some point, they would have to release their cargo and return to an earlier point to repeat the process. Receptor models have, in the past, suffered from the lack of obvious candidates for the receptors. The realization, however, that a number of membrane proteins are at least capable of cycling between ER and Golgi has changed this situation. The CGN proteins such as p53 and p58 could, conceivably, help to concentrate secretory proteins. Such possibilities are not easy to test but, given the apparent paradox of unidirectional transport along the secretory pathway, they are worth considering. The existence of recycling membrane proteins, whether or not they act as receptors, opens a new set of questions.

Recycling

of proteins

between

the endoplasmic

reticulum

0

Fig. 2. Three ways in which rapidly transported proteins could be concentrated. (a) Bulk flow. Proteins leave the endoplasmic reticulum (ER) by massive nonselective vesicular transport fc3), and travel onwards by a similar mechanism, but more slowly. Excess lipid is returned to the ER by a hypothetical non-vesicular route (:::3; retrieval of some proteins occurs by a separate, selective vesicular pathway f+). fb) Trapping. initial transport is by bulk flow, and excess lipid returns to the ER by vesicular transport f-j. Secretory proteins are prevented from returning by interaction with hypothetical receptors in the cis-Golgi network KCN); as CCN elements move to the Golgi region and mature, the receptors release their cargo and are then selectively recycled (possibly, but not necessarily, through the ER). fc) Receptormediated transport. Proteins bind to hypothetical receptors in the ER, and are transported by a relatively small number of vesicles to the CCN. Other proteins would be carried non-selectively in the same vesicles. The receptors subsequently have to drop their cargo and return to the ER.

Such proteins must bear derings, but we have little of these signals or about them. There are still many

signals that dictate their waninformation about the identity the machinery that recognizes sorting problems to be solved.

5. .

and recommended

Papers of special interest, published have been highlighted as: . of interest .. of outstanding interest MLINRO

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Genetic selection, cloning, and analysis of a yeast gene that affects the HDEL retention system. Shows that the Golgi complex is involved in the sorting of ER proteins, and that growth conditions can have a significant effect on the retrieval pathway.

7. .

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HRB:

Analysis of the carbohydrate mod&ations that occur when yeast se cretory proteins are tagged with HDEL Together with the use of Set mutants and subcellular fractionation, this provides the best evidence for recycling of ER proteins through the Golgi apparatus. 6.

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Shows that the yeast, K kauis, has DDEL at the carboxyi ~errninus of one ER protein (BiP), and that S cerevi&e does not e5icientIy recognize this as a retention signaI unless its ERD2 gene is replaced by the K hcfis equivalmt Together with [7e], this provides the best evidence that the ERD2 gene product is the HDEL receptor.

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591