The retention signal for soluble proteins of the endoplasmic reticulum

TIBS15 - DECEMBER1990 Hanecak, R., Duprey, E. and Wimmer, E. (1984) J. Virol. 50, 507-514 18 Sangar, D. V., Newton, S. E., Rowlands, D. J. and Clarke, B. E. (1987) Nucleic Acids Res. 15,

3305-3315 19 Palmenberg,A. C., Kirby, E. M., Janda, M. R.,

Drake, N. L., Duke, G. M., Potratz, K. F. and Collett, M. S. (1984) Nucleic Acids Res. 12, 2969-2985 20 Howell, M. T., Kaminski, A. and Jackson, R. J. (1990) in New Aspects of Positive Strand RNA Viruses (Brinton, M. and Heinz, F. Z., eds), pp. 144-151, American Society for Microbiology 21 Kaminski, A., Howell, M. T. and Jackson, R. J. (1990) EMBO J. 9, 3753-3759 22 Pelletier, J., Kaplan, G., Racaniello, V. R. and

Sonenberg, N. (1988) Mol. Cell. BioL 8, 1103-1112 23 Bienkowska-Szewczyk,K. and Ehrenfeld, E. (1988) J. Virol. 62, 3068-3072 24 Kuge, S., Kawamura, N. and Nomoto, A. (1988) J. ViroL 63, 1069-1075 25 Ilzuka, N., Kohara, M., Hagino-Yamagishi,K., Abe, S., Komatsu, T., Tago, K., Arita, M. and Homoto, A. (1989) J. Virol. 63, 5354-5363 26 Staehelin, T., Trachsel, H., Erni, B., Boschetti, A. and Schreier, M. H. (1975) FEBS Symp. 39, 309-323 27 Meerovitch, K., Pelletier, J. and Sonenberg, N. (1989) Genes Dev. 3, 1026-1034 28 del Angel, R. M., Papavassiliou, A. G., Fernandez-Thomas,C., Silverstein, S. and

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SECRETION OF PROTEINS from eukaryotic cells is a complex process. Newly synthesized secretory and membrane proteins enter the endoplasmic reticulum (ER) in an unfolded state, where they undergo modifications such as glycosylation, disulphide bond formation and assembly into oligomers. They are then transported through a series of membrane-bound compartments which include the various cisternae of the Golgi complex, where further carbohydrate modifications occur (for reviews, see R e f s 1,2). Transport between compartments occurs by means of vesicles that bud and fuse in a specific manner; once within the secretory pathway, proteins do not have to cross a membrane to reach the cell surface. The complexity of this system has certain advantages for the cell, because it allows proteins to fold and mature in closed compartments that contain appropriate enzymic catalysts of these processes. However, its successful operation is dependent on sorting mechanisms that position the enzymes correctly and maintain them in place despite the continual flow of material through the secretory pathway. The ER presents a particularly striking sorting problem. In recent years it has become clear that this organelle contains a number of abundant soluble proteins that aid the initial steps in the maturation of secretory proteins 2. These include BiP (binding protein, a homologue of the 70 kDa heat-shock protein), which is involved in the H. R. B. Pelham is at the MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.

Racaniello, V. R. (1989) Proc. Natl Acad. Sci. USA 86, 8299-8303 29 Najita, L. and Samow, P. (1990) Proc. Natl Acad. Sci. USA 87, 5846-5850 30 Earle, J. A. P., Skuce, R. A., Fleming, C. S., Hoey, E. M. and Martin, S. J. (1988) J. Gen. Virol. 69, 253-263 31 Munoz, A., Alonso, M. A. and Carrasco, L. (1984) J. ViroL 137, 150-159 32 Dolph, P. J., Racaniello, V. R., Villamarin, A., Palladino, F. and Schneider, R. J. (1988) J. Virol. 62, 2059-2066 33 Dolph, P. J., Huang, J. and Schneider, R. J. (1990) J. Virol. 64, 2669-2677 34 Sarnow, P. (1989) Proc. Natl Acad. Sci. USA 86, 5795-5799

!

The lumen of the endoplasmic reticulum (ER) contains a number of soluble proteins, many of which help the maturation of newly synthesized secretory proteins. Retention of these resident proteins in the ER is dependent on a carboxy-terminal signal, which in animal cells is usually Lys-Asp-Glu-Leu (KDEL). This signal is thought to be recognized by a membrane-bound receptor that continually retrieves the proteins from a later compartment of the secretory pathway and returns them to the ER. COS cells, a truncated form of BiP that lacked this sequence was secreted 4. Similar experiments have demonstrated the importance of carboxy-terminal sequences for the retention of PD! and a 72 kDa protein (ERp72) in mammalian cells 5, and of BiP in Saccharomyces cerevisiaeE Conversely, addition of the last six amino acids of BiP to secretory, lysosomal or vacuolar proteins caused these proteins to accumulate in the ER of animal cells 4,7-~°, yeast H,~2 and plants ~3,~4.Thus, retention of luminal ER proteins is a specific, signal-dependent phenomenon. The demonstration of a similar retention system in widely divergent species suggests that it is a universal feature of eukaryotes. The requirements for a functional signal can be deduced in two ways: by The ER retention signal A common carboxy-terminal tetra- comparing the sequences of known ER peptide, KDEL, was first noticed when residents, and by testing artificial the sequences of rat BiP and PD! were sequences for retention activity. From compared3. The same sequence (or a sequence comparisons, a number of closely related one) was subsequently generalizations have emerged2. The sigfound at the carboxyl terminus of other nal is always found at the extreme carluminal ER proteins from a number of boxyl terminus, and there is no evispecies 2. When expressed in monkey dence for conservation of more than

import and folding of proteins, protein disulphide isomerase (PDI), which is required for correct disulphide bond formation, and GRP94 (a glucose-regulated protein, also known as endoplasmin), which is a homologue of the 90 kDa heat-shock protein. Another abundant ER protein is calreticulin, whose only known property is its ability to bind calcium. These proteins are resident in the ER, and must somehow be distinguished from newly synthesized secretory proteins, which are rapidly transported to the Golgi apparatus. Subsequent transport steps present fewer sorting problems since Golgi cisternae seem to lack soluble resident proteins.

© ]990,E|sevierSciencePublishersLtd, (UK) 0376-5067/90/$02.00

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TIBS1 5 - DECEMBER1990 Table I. Carboxy-termlnal sequences of luminal ER proteins a equence

Protein

Species

DEL

BiP

rat, mouse, hamster, human, chick, Drosophila melanogaster, Caenorhabditis elegans (gene 1) mouse, hamster, human 25, chick rat, mouse, cow, human, chick rat26, mouse27, rabbit28 C. elegans maize29

GRP94 PDI calreticulin gut esterasec auxin binding protein DEL

DEL DEL DEL DEL EEL ~EDL IEL TEL QDL

BiP GRP94b PDI Kre5pc

tomato, maize13, C. elegans (gene 2), Saccharomyces cerevisiae S. cerevisiae 6, Kluyveromyces lactis21 S. cerevisiae a S. cerevisiae 15

BiP BiP BiP colligin ERp72 PI-PLC form ic,d, a liver esterase a liver esterase a PDl-like proteinc,d

K. lactis21 Schizosaccharomyces pombea Plasmodium falciparum rat mouse5 rat3° rabbit rabbit Trypanosoma brucei 31

References not given here can be found in Ref. 2. The S. pombe BiP and S. cerevisiae PDI sequences re unpublished observations of A. Pidoux and J. Armstrong, and M. Tuite and R. Freedman, respectively. The GRP94 homologues of S. cerevisiae and K. lactis were tentatively identified as glycoproteins of le appropriate size, and their carboxy-terminal sequences deduced from their reactivity with anti-HDEL ntibodies. These proteins have not been shown to be resident in the ER. Their assignment as such is based olely on their possession of a signal peptide and a KDEL-like sequence. The reported sequence of a rat phosphoinositol-specific phospholipase C (PI-PLC) shows homology with DI. Many of the homologous residues are also found in the T. brucei protein.

efficiency of retention can vary, depending on the test protein and the linker between the protein and the retention signal. Presumably this reflects variations in the accessibility of the signal in different configurations8,~,~3. The primary role of the KDEL system appears to be the retention of soluble ER proteins, rather than membrane proteins. Some membrane proteins are thought to be kept in place by their association with large aggregates that cannot enter transport vesicles 2. Others, characterized by a single transmembrane domain and a short carboxyterminal cytoplasmic tail, carry a cytoplasmic retention signal TM. Recently, we have identified the first example of a membrane protein with an HDEL signal in S. cerevisiae. This is the product of the SEC20 gene, which is predicted to have its amino terminus in the cytoplasm, a single transmembrane segment, and a luminal sequence that terminates with HDEL (D. Sweet, unpublished). At present we do not know the precise location of this protein, but since it is required for ER-to-Golgi transport ~9, it is likely that it is a substrate for the HDEL retention system. Retention by retrieval

e terminal four amino acids. The pre- cells are the most striking exceptions. ;e sequence of the signal varies Some of these have divergent but recog~tween species, but is usually very nizable signals (HIEL, HTEL), but other 'ictly conserved on the major ER pro- related members of the family have ins. However, less abundant proteins completely different carboxyl termini metimes have divergent sequences. It (e.g. TEHT, see Ref. 16). It remains possible that efficient retention of unclear whether these proteins are ese proteins is not crucial for the cell; retained by the same system. Other deed, even ER proteins that lack the exceptions may have trivial explarboxy-terminal signal are secreted nations. For example, the enzyme prolyl ]y slowly, and removal of the signal 4-hydroxylase has two different sub~m yeast BiP or Kre5 protein (an units, one of which is identical to PDI zyme involved in cell wall synthesis) while the other subunit lacks a KDEL ,es not have lethal consequences 6,15. signal 17, but the enzyme is presumably Table I shows some examples of retained via its association with PDI. rboxy-terminal sequences. In verThe general features of the mam~rates, KDEL seems to be the pre- malian retention signal have been •red signal. Several plants use HDEL, confirmed by experimental manipuhough KDEL is also found; both lation (see Table II for summary and refquences are also used in the nema- erences). Thus, addition of amino acids de Caenorhabditis elegans. Among the to the carboxyl terminus abolishes asts there is considerable variation: retention. Some changes to the first two cerevisiae uses HDEL, the related amino acids are tolerated, including the dding yeast Kluyveromyces lactis net loss or gain of one charge; for ex:ognizes both HDEL and DDEL, while ample, KNEL and DKEL are functional, from the fission yeast Schizo- although DDEL is not. Alteration of the ccharomyces pombe has ADEL. other residues has a more drastic effect riant sequences generally have con- - even the removal of a single CH2 rvative changes (R for K, D for E), but group from the terminal leucine (conecise conservation of the net charge verting it to valine) is sufficient to aboles not seem to be essential. The ER ish function. One other conclusion has terases found in mammalian liver come from these experiments: the

How does the retention process work? In principle, KDEL-tagged proteins could be immobilized by their attachment to a receptor in the ER, and thus be unable to enter budding transport vesicles. However, there is considerable evidence that the luminal ER proteins are soluble, and that the presence of a KDEL sequence does not alter their diffusion rate within the ER2. Rather, it seems that the proteins are able to leave the ER, but are promptly retrieved from a later compartment and returned to their original position. This can be demonstrated by examining the carbohydrate modifications that occur to proteins to which a retention signal has been added. Thus in animal cells, addition of KDEL to the lysosomal enzyme cathepsin D causes this enzyme to accumulate in the ER, but does not prevent the addition of GlcNAc-l-phosphate, a modification that is thought to occur in a post-ER compartment 1,7. Similarly, HDEL-bearing fusion proteins expressed in S. cerevisiae undergo an early Golgi modification, but are found in the ER; in this case, it is easy to prove that the modifications occur in a compartment topologically distinct from the ER, because they do not occur (at the non-permissive temperature) in

TIBS15-DECEMBER1990

a temperature-sensitive mutant that is defective for vesicular transport ~2. Such results have led to a simple model for retention. Secretory proteins are transported in a non-specific manner to a post-ER compartment, and some resident ER proteins inevitably get carried along with them. In this compartment, some as yet unknown feature of the environment promotes binding of the KDEL signal to a receptor which is an integral membrane protein. The receptor-ligand complexes are then incorporated into a special class of vesicles that return to the ER where the ER proteins are released. The receptor can return to the salvage compartment in the general flow of secretory proteins, to await the passing of the next KDEL-bearing molecule. The SEC20 membrane protein presumably travels the same route, and may be involved in one of the vesicular transport steps of this pathway. Many important details of the retrieval process remain to be elucidated, but the recent identification of the sorting receptor has lent substance to this model, and should provide a starting point for more detailed investigation.

Table II. Mutant signals tested In mammalian cells Signals that work

Signals that do not work

RDEL1° KNEL10 DKEL1°

KDELGL4 HDEL11 DDELa KDAS4 KDQL1° KDEA10 KDEV8

a M. Lewis, unpublished.

implying that it is the receptor. Moreover, the single K. lactis receptor must have dual specificity. The S. cerevisiae receptor is 219 amino acids in length 2°, and is predicted to have seven transmembrane domains. It is likely that the HDEL binding site is in a pocket formed by two or more of these domains, and analysis of chimeric K. lactis-S, cerevisiae ERD2 molecules has shown that the sequences that confer the ability to recognize DDEL On addition to HDEL) can be localized to the luminal end of one of the transmembrane segments (J. Semenza, unpublished). Immunofluorescence and subcellular fractionation studies indicate The sorting receptor that the majority of the receptor is Because it was hard to predict the found in a post-ER, Golgi-like compartproperties of the receptor in advance, ment, consistent with the recycling we chose a genetic approach to its model outlined above. The use of oligonucleotides correidentification. Mutants of S. cerevisiae were isolated that were unable to retain sponding to conserved regions of ERD2 fusion proteins bearing the HDEL signal; as primers in the polymerase chain two genes were identified in this way, reaction has allowed the isolation of an termed ERDI and ERD2 (for ER reten- ERD2 homologue from human cells which encodes a protein that is 50% tion defective)6,u,2°. Mutations in ERDI affect Golgi function, and may block the identical to the S. cerevisiae receptor 22, retrieval of ER proteins indirectlyE The and so is very likely to be the KDEL ERD2 gene encodes a membrane pro- receptor. Unfortunately, attempts to tein with properties that are expected express the human protein in yeast for the receptor - in particular, the level cells to confirm its activity have not yet of its expression controls the capacity been successful. It has, however, been of the retention system, which is quite expressed in monkey COS cells; its easily saturated in S. cerevisiae 2°. product was found primarily in the Evidence that the ERD2 product recog- vicinity of the Golgi apparatus, but was nizes the retention signal came from a also observed in dispersed vesicular more elaborate experiment. As men- structures that may be intermediates tioned above, the budding yeast K. lac- between ER and Golg£2. An alternative approach has also tis uses both HDEL and DDEL as retention signals, but testing of the DDEL been used to identify a candidate for sequence in S. cerevisiae showed that it the mammalian KDEL receptor. Antiwas very poorly recognized. When the bodies were prepared against KDELS. cerevisiae ERD2 gene was replaced containing peptides, and monocional by the equivalent gene from K. lactis, anti-idiotypic antibodies were isolated; proteins with HDEL or DDEL were these antibodies, which in principle retained with equal efficiency, although should have some features in common KDEL proteins were still secreted2L with the KDEL sequence itself, recogThus, the ERD2 product determines the nized a 72 kDa glycoprotein on western specificity of the retention system, blots 23. This protein was localized in an

intermediate compartment between ER and Golgi, and also showed a weak affinity for KDEL sequences. However, it is clearly different from the 25 kDa ERD2 homologue referred to above. Thus the 72 kDa protein has several properties expected for a receptor, but in the absence of genetic data it is difficult to prove that it has a role in the retention process. Cloning and subsequent manipulation of the gene that encodes it may help to resolve this uncertainty. Conclusions The presence of KDEL or a closelyrelated sequence at the carboxyl terminus of a protein that also carries an amino-terminal signal sequence is a very strong indicator that the protein is normally a resident of the ER lumen. The basic retention system for such proteins must have evolved early in eukaryote history, and has been well conserved, although slight differences in the signal have arisen in different species. This drifting of specificity was probably facilitated by the relatively small number of proteins which require sorting, and whose sequences thus have to remain compatible with the receptor specificity. Many questions about the retention system have yet to be answered. For example, it is not clear how the receptor itself is prevented from passing too far down the secretory pathway. We do not know how the binding and release of KDEL is regulated, nor whether the sequence of the retention signal arose by chance, or whether it has evolved to aid the sensing of signals (such as ion concentration or pH) that control receptor binding. In some cells, calcium ionophores have been shown to cause secretion of ER proteins, raising the possibility that calcium is involved in the binding or release of KDEL. However, many cells do not show this response, and it may reflect a more general disruption of ER structure and functionz4. The identification of the receptor should make possible the biochemical studies that will be required to resolve such questions. References 1 Kornfeld, R. and Kornfeld, S. (1985) Annu. Rev. Biochem. 54, 631-634 2 Pelham, H. R. B. (1989) Annu. Rev. Cell. Biol. 5, 1-23 3 Munro, S. and Pelham, H. R. B. (1986) Cell 46, 291-300 4 Munro, S. and Pelham, H. R. B. (1987) Ce1148, 899-907

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TIBS15-DECEMBER1990 5 Mazzarella, R. A., Srinivasan, M., Haugejorden, S. M. and Green, M. (1990) J. Biol. Chem. 265, 1094-1101 6 Hardwick, K. G., Lewis, M. J., Semenza,J., Dean, N. and Pelham, H. R. B. (1990) EMBO J. 9, 623-630 7 Pelham, H. R. B. (1988) EMBO J. 7,913-918 8 Zagouras, P. and Rose, J. K. (1989) J. Ceil Biol. 109, 2633-2640 9 Buonocore, L. and Rose, J. K. (1990) Nature 345, 625-628 10 Andres, D. A., Dickerson, I. M. and Dixon, J. E. (1990) J. Biol. Chem. 265, 5952-5955 11 Pelham, H. R. B., Hardwick, K. G. and Lewis, M. J. (1988) EMBO .I. 7, 1757-1762 12 Dean, N. and Pelham, H. R. B. (1990) J. Cell Biol. 111, 369-377 13 Chrispeels, M. J. Annu. Rev. Plant Physiol. Plant Mo/. Biol. (in press) 14 Herman, E. M., Tague, B. W., Hoffman, L. M., Kjemtrup, S. E. and Chdspeels, M. J. P/anta (in press)

T H E BIOSYNTHESIS OF the modified tetrapyrroles has attracted the attention and imagination of chemists and biochemists alike for the past fifty years. This class of compounds includes the metallopigments haem (the prosthetic group of haemoproteins such as haemoglobin, cytochromes and catalase) sirohaem (the prosthetic group of sulphite and nitrite reductases) chlorophyll (the biological solar energy molecule) factor F430(the cofactor of the enzyme methyl CoM reductase) and vitamin Blz (the cofactor of several methylation and rearrangement reactions and, in evolutionary terms, the matriarch of all the tetrapyrrolic derived macrocycles). The fascination of those molecules stems from their structural complexity - Vitamin B~2 has been described as second only in structural complexity to DNA and proteins and from their derivation from one unsymmetrical isomer of a hexahydroporphyrin, uroporphyrinogen IlI (Fig. 1). The natural modification of the conjugation state and ring size of these tetrapyrroles has permitted the coordination of different metals at the ring centre and, together with the variations in the electronic configuration, has endowed a wide range of different biological prosthetic functions for uroporphinoids.

M. J. Warren and A. I. ,Scott are at the Center for Biological NMR, Department of Chemistry, Texas A and M University, College Station, TX 77843-3255, USA.

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15 Meaden, P., Hill, K., Wagner, J., Slipitez, D., Sommer, S. S. and Bussey, H. (1990) Mo/. Cell. Biol. 10, 3013-3019

16 Long, R. M., Satoh, H., Martin, B. M., Kimura, S., Gonzalez,F. J. and Pohl, L. R. (1988) Biochem. Biophys. Res. Commun. 156, 866-873 / 7 Helaakoski, T., Vuori, K., Myllyl~, R., Kivirikko, K. I. and Pihlajaniemi, T. (1989) Prec. Nat/ Acad. Sci. USA 86, 4392-4396 18 Jackson, M. R., Nilsson, T. and Peterson, P. A. (1990) EMBO J. 9, 3153-3162 19 Kaiser, C. A. and Scheckman, R. (1990) Ceil61, 723-733 20 Semenza,J. C., Hardwick, K. G., Dean, N. and Pelham, H. R. B. (1990) ce//61, 1349-1357 21 Lewis, M. J., Sweet, D. J. and Pelham, H. R. B. (1990) Ceil61, 1359-1363 22 Lewis, M. J. and Pelham, H. R. B. (1990) Nature (in press) 23 Vaux, D., Tooze,J. and Fuller, S. (1990) Nature

345, 495-502 24 Booth, C. and Koch, G. L. E. (1989) Cell 59,

729-737 25 Maki, R. G., Old, L. J. and Srivastava, P. K. (1990) Proc. Natl Acad. Sci. USA 87,

5658-5662 26 Murthy, K. K., Banville, D., Srikant, C. B.,

Carrier, F., Holmes, C., Bell, A. and Patel, Y. C. (1990) Nucleic Acids Res. 18, 4933 27 Smith, M. J. and Koch, G. L. E. (1989) EMBO J. 8, 3581-3586 28 Riegel, L., Burns, K., MacLennan,D. H., Reithmeier, R. A. F. and Michalak, M. (1989) J. Biol. Chem. 264, 21522-21528 29 Inhohara, N., Shimomura, S., Fukui, T. and Futai, M. (1989) Prec. Natl Acad. Sci. USA 86, 3564-3568 30 Bennett, C. F., Balcarek, J. M., Varrichio, A. and Crooke, S. T. (1988) Nature 334, 268-270 31 Hsu, M. P., Muhich, M. L. and Boothroyd,J. C. (1989) Biochemistry 28, 6440-6446

Data obtained using a combination of molecular biology and NMR spectroscopy has transformed our thinking about the evolution of the biochemical machinery required for the synthesis of the vital metallopigments: haem, chlorophyll, vitamin B12 and factor F43o. One of the most recent advances is the discovery of a unique dipyrromethane cofactor that is bound covalently at the active site of porphobilinogen deaminase, the key enzyme of tetrapyrrole assembly. We will also discuss how the oxidation level and chromophoric arrangement of the uroporphinoid ring, rather than its substitution pattern, provides the necessary molecular recognition for some of the later enzymes, whose function is to decorate the template by C-methylation on the way to the biologically active cofactors.

The first committed steps towards the biosynthesisof tetrapyrrolos The first committed precursor in the tetrapyrrole biosynthetic pathway is 5aminolaevulinic acid (ALA). In mammals and photosynthetic bacteria, this aminoketo acid is synthesized from succinyl CoA and glycine by the pyridoxal phosphate-requiring enzyme ALA synthase, along the 'Shemin' route 1 mediated by the hemA gene. However, in higher plants and many prokaryotic systems, ALA is synthesized from the intact carbon skeleton of glutamate using the C-5 pathway2, which is an interesting transformation as it is one of the few reactions that utilizes a tRNA

molecule in a process other than protein biosynthesis (Fig. 2). Two molecules of ALA are then condensed in a Knorr-type reaction by the enzyme ALA dehydratase, encoded by hemB, to yield porphobilinogen (PBG) (Fig. 2), the building block of tetrapyrrole biosynthesis. A detailed study of this enzyme has shown it to comprise eight identical subunits and to require a minimum of four zinc molecules. The first ALA molecule binds to the enzyme through a lysine group and this ALA molecule ends up as the right hand side of the final pyrrole, thereby forming the propionate side chain moiety, while the acetate side chain occupies the

© 1990,ElsevierSciencePublishersLtd,(UK) 0376-5067/90/$02.00