Heterologous expression of the red-cell anion exchanger (band 3; AEI)

Expression and Purification of Membrane Proteins

I8 Poolrnan, B., Molenaar, D. and Konings, W. N. (I 994) in Handbook of Biomernbranes, vol. 2 (Shinitzky, M., ed.), pp. 329-379, Balaban Publisher;, Rehovot

15 Rigaud, 1.-L,Patemostre, M.-T. and Bluzat, A. ( I 988) Biochemistry 27,2677-2688 16 Kragh-Hansen, U., Le Maire, M. and Mdler, J.V. ( I 998) Biophys. J. 75,2932-2946 17 Fang, G., Friesen, R M. E., Lanfenneijer, F., Hagting, A,, Poolman, B. and Konings, W. N. (I 999) Mol. Mernbr. Biol.,

Received I9 May I999

in the press

Heterologous expression of the red-cell anion exchanger (band 3; AE I ) Jonathan D. Groves*', Mark D. Parker*, David Askin*,Pierre Falsont, Marc le Mairet and Michael j. A. Tanner* *Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 ITD, U.K., and tDepartement de Biologie Cellulaire et Moleculaire, CEA et CNRS URA 2096, CEA de Saclay, F-9 I I9 I Gif-sur-Yvette, France

3 molecules were expressed in a functional form at the cell surface. T h e development of heterologous expression systems for functional band 3 is considered to be of importance for several reasons. First, to provide a simple means for study of the anion-exchange mechanism and structure-function relationship of band 3, using site-directed mutagenesis. Second, to over-produce sufficient quantities of correctly folded homogeneous material for structural studies to be undertaken with native or recombinant mutant forms of band 3. Third, to delineate specific interactions, either between elements of secondary structure within the band 3 dimer, or between band 3 and other red-cell plasma membrane or cytoskeletal proteins with which it forms complexes. Fourth, to investigate how the biosynthesis of band 3 is regulated to avoid the potential problems associated with a pHmodifying transporter in intracellular membranes.

Introduction T h e human red-cell anion exchanger (band 3 ; AE1) has been widely used as a model protein for the study of membrane transport due to its high abundance in the erythrocyte membrane (1.2 x lo6 copies/cell; [l]). Band 3 is a multifunctional integral protein that comprises two distinct structural and functional domains (reviewed in [2-4]) : the 43-kDa N-terminal cytoplasmic domain (residues 1-359) is involved in binding of the red-cell cytoskeleton and other functions, while the 52kDa C-terminal membrane domain (residues 36091 1) is both necessary and sufficient for mediation of the chloride-bicarbonate-exchange function. T h e membrane domain traverses the bilayer either 12 or 14 times [5] and contains a substantial proportion of a-helical structure. Human band 3 is almost exclusively oligomeric in the red-cell membrane, and the membrane domain has been reconstituted as a dimer with lipids to form twodimensional crystals which have yielded a lowresolution structural model [6]. Erythroid band 3 cDNAs have been cloned from mammals (mouse, rat, human), birds (chicken) and fish (trout). T h e primary sequence of the anion exchanger is highly conserved between these species, with the greatest homology in the putative transmembrane spans (reviewed in [7]). Successful expression of red-cell band 3 has been obtained using a variety of eukaryotic expression systems, including Xenopus oocytes, insect cells, yeast and tissue-culture cells (Table 1). In the majority of cases, a proportion of the band

Expression of band 3 in Xenopus oocytes T h e heterologous expression of erythroid band 3 was first demonstrated by Passow and co-workers [8], who injected Xenopus oocytes with sizefractionated poly(A)+ mRNA from anaemic mice. They showed a 24-fold increase in uptake of radiolabelled chloride into injected cells compared with control cells, and that the enhanced transport activity was sensitive to stilbene disulphonate band 3 inhibitors. They also demonstrated the biosynthesis of band 3 by immunoprecipitation with polyclonal mouse anti-band 3 antibodies. Following the successful cloning of the mouse band 3 cDNA [9], cRNA was transcribed in vitro from an upstream SP6 RNA polymerase promoter

Abbreviations used: GPA, glycophorin A ; b3mem, integral rnernbrane domain of band 3. 'To whom correspondence should be addressed.

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Intact cells Not detected with intact cells

Xenopus oocytes

5. cerevisiae (constitutive)

5. cerevisiae (inducible)

5. cerevisiae (inducible)

Human band 3 cDNA (183-91 I)

Human band 3 cDNA (361-91 I) Human band 3 cDNA (1-91 I)

HEK293 cells (inducible)

6 6 2 cells (stable)

Human band 3 cDNA

Human band 3 cDNA

*

HEK293 cells (inducible)

Human band 3 cDNA

The mouse band 3 clone used in this study [34] may have contained point mutations that affected intracellular targeting [2 I].

Intact cells

47% of total band 3 350 dpm’

99- and 102-kDa isoforms only 8.5 x 10’ dcell [x 14% of total band 31

HEL cells (transient) Intact cells

Detected

Estimated I I % of total 5.7 x Io4 dcell 56 dpm’ None detected

20 dpm’ None detected

5- I 0% of total band 3

IC250 cells (transient)

Proteoliposomes and microsomes

Intact cells

Intact cells

Intact cells

I .2 x Io6 dcell 7000 dpm’ ( I -2) x I 0’ dcell 2-3 dpm’ 20-40 dpm’ [ 1-2 x I 0’ dce111 Assumed 10% of total [ I0’- Io9 dce111 [20-200 dpm2] Anion channel and exchanger None detected

Cell-surface expression

Mouse band 3 cDNA (I -929)* Mouse kidney band 3 cDNA (8C-929) Chicken band 3 cDNA

HEK293 cells (transient)

Proteoliposomes only

Xenopus oocytes Xenopus oocytes

Human band 3 cRNA Human band 3 cRNA (1-91 I and 361-91 I) Trout band 3 cRNA

Baculovim Sf-9

Intact cells

Xenopus oocytes

Mouse band 3 cRNA

Human band 3 cDNA

Intact cells Intact cells

Xenopus oocytes

Mouse poly (A)’ mRNA

Endogenous

Erythrocytes

Native human band 3

Functional expression

Expression system

Ongin of band 3

[6.4 x 10’ dcell]

I pg/106 cells [6 x lo6 dcell]

[0.08 my1 of cutture] 5 x 10 dcell 0.23 % total protein

0.7 mg/l of cutture I.5 % membrane proteins 0.1 mg/l of culture 2 x Io4 dcell

2.3 x 10’ dcell

Total expression

Some undefined glycosylation High mannose only Possibly limited glycosylation

High mannose only

Unglycosylated

Unglycosylated

Unglycosylated

Unglycosylated

High mannose only

Complex polylactosaminyl

N-glycosylation

~

~~

PI

(unpublished)

J. D. Groves and M. J. A. Tanner

r321

Reference

~

3. E

Results indicated in square brackets have been calculated from data in the references shown. The average diameter of an oocyte is assumed to be I.2 mm; the surface area of an oocyte is assumed to be x 4.5 pm2,which is 2-fold higher than the calculated value to allow for microvilli in the plasma membrane [8]. dcell, copiedcell; dpm2, copiedpm2.

2

C

< o_

-3

8

v,

n

Heterologous expression of the red-cell anion exchanger (band 3) in eukaryotic systems

(D

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and injected into oocytes [lo]. This approach resulted in expression of significantly higher levels of functional band 3 in the plasma membrane than using purified mRNA. In subsequent studies, these workers have measured both chloride uptake into groups of oocytes and efflux from single cells using a range of normal and mutagenized mouse band 3 cDNA constructs (e.g. [l 1-1 31). This work has yielded information about the anion-transport pathway in mouse band 3 and has established that the kinetic properties of band 3 expressed in oocytes are similar to those in red cells. The human band 3 cDNA [14] has also been expressed in the plasma membrane of Xenopus oocytes [15-171 where it becomes core N-glycosylated, although the glycan is not processed further [18]. The proportion of band 3 expressed at the cell surface has been quantified by treatment of intact oocytes with chymotrypsin, papain or pronase [16,191, which cleaves exposed exofacial sites of the cell-surface band 3 population. From the work conducted in our laboratory, we have identified two key factors which greatly facilitate cell-surface expression. First, by increasing the quantity of expressed band 3 in the oocyte membranes, thereby favouring oligomerization, which is a concentration-dependent process. We achieved this (i) by subcloning the band 3 cDNA into the Bluescript-based plasmid vector pBSXGl [16], which contains 5’ and 3’ untranslated flanking regions of the Xenopus B-globin cDNA together with a poly(A)+ tail, and (ii) by injecting 7-methyldiguanosine triphosphate (m’GpppG) capped band 3 cRNA (mMessage mMachine@, Ambion) at increased concentrations ( > 1 5 ng/ oocyte). Second, by co-injecting the cRNA encoding red-cell glycophorin A (GPA), a singlespanning membrane protein that facilitates the movement of band 3 to the oocyte plasma membrane and has been shown to interact with band 3 in native red cells (reviewed in [20]). Subsequently, the Xenopus oocyte system has been exploited to investigate the structure-function, cell-surface targeting and biosynthetic processes of a variety of human band 3 mutants. Studies using engineered recombinant forms have included : (i) site-directed substitution of specific amino acid residues such as the endogenous Nglycosylation site at Asn-642, which was found not to be essential for function, at least in the presence of GPA [18]; (ii) deletion of parts of the Cterminus of mouse [21] or human [22] band 3, which resulted in loss of cell-surface expression; and (iii) co-expression of two or more comp-

lementary truncated fragment portions of band 3, which showed that division in any of six different loops or deletion of two entire transmembrane spans does not abolish function, and that there are multiple signals for insertion and assembly of band 3 in the membrane [22-251. An N-terminally truncated isoform of erythroid band 3 is also expressed in the intercalating cells of the distal tubule of the kidney. Mouse kidney band 3 (which lacks residues 1-79 of mouse band 3) and human kidney band 3 (which lacks residues 1-65 of human band 3) have been functionally expressed in oocytes [26,27]. The expression of several naturally occurring human variant forms of either erythroid or kidney band 3 has been examined in oocytes, including the variants responsible for South-east Asian ovalocytosis [28], band 3 Prague [19] and familial distal renal tubular acidosis [27]. The oocyte system has also been used for functional expression of trout band 3. FiCvet and co-workers [29] showed that trout band 3 possessed both chloride exchange and channel activities, in contrast with mouse band 3, which mediated only anion exchange. In addition, trout band 3 uniquely elicited transport of the organic solute taurine, which has been implicated in cellvolume regulation.

Expression of band 3 in yeast The yeast Saccharomyces cerevisiae has been used extensively for heterologous gene expression, although only a limited number of integral membrane proteins have been expressed functionally in this system (reviewed in [30]). It appears that in general, mammalian membrane proteins are expressed in yeast less readily than their plant or fungal equivalents and in many cases accumulate in perinuclear membranes. Sekler and co-workers [31] reported the first expression of a band 3 recombinant (containing ten amino acid residues of phosphoglycerate kinase, a six-histidine residue affinity tag and amino acid residues 183-911 of human band 3) in yeast using a constitutive promoter. Although the levels of expression were reasonably high (Table l ) , the expressed band 3 was localized to intracellular membranes and required partial purification and reconstitution into proteoliposomes to confirm functionality. In a collaboration between the authors’ two laboratories, we have expressed the integral membrane-domain portion of human band 3 (b3mem; residues 361-91 1 ) in a pep4 protease-defective strain of S . cerevisiae under the control of a

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Biochemical Society Transactions ( I 999) Volume 27, part 6

We co-transformed yeast with two independent vectors containing cDNAs encoding b3mem and red-cell GPA. A double complementation procedure was used to select for cells containing both plasmids. T h e GPA polypeptide was expressed from a constitutive promoter and b3mem expression was induced with galactose (as described above). Stilbene disulphonate-sensitive chloride uptake and exofacial cleavage of cellsurface b3mem with chymotrypsin were both enhanced by co-expressed GPA (M. D . Parker, J. D . Groves and M. J. A. Tanner, unpublished work). This result parallels the effect of coexpressed GPA on b3mem in Xenopus oocytes, and demonstrates that the effect is not specific to the oocyte system. T h e chloride-bicarbonate-exchange function of band 3 has the potential to modify the p H of intracellular compartments of the yeast cell. We compared the expression of intact band 3 and b3mem with that of two non-functional mutants (the Glu681 + Gln point substitution and the South-east Asian ovalocytosis deletion of residues 4 0 M 0 8 ) ; in each case, the expression of the wildtype and mutant constructs and yeast cell growth rates were similar (J. D . Groves, P. Falson, M . le Maire and M. J. A. Tanner, unpublished work). This indicates that the amount of expressed polypeptide is not limited by band 3-mediated anion transport. We also examined the expression of b3mem in yeast cells which were cultured in the presence or absence of 5 0 m M NaCl (J. D. Groves, D. Askin and M. J. A. Tanner, unpublished work). On immunoblots, we found that the proteolytic cleavage and carboxypeptidase Y digestion of b3mem were considerably lower if the cells were grown in medium containing chloride, while cell doubling times and stilbene disulphonate-sensitive chloride-uptake rates were not affected. It is clear that there are a number of different intracellular targeting pathways for band 3 in yeast. These results indicate that a very wide range of constructs, host strains and growth parameters should be screened at an early stage in the development of a membrane-protein overexpression system in yeast, especially when studying a physiologically active membrane protein such as band 3.

galactose-inducible promoter [32]. Unexpectedly, we found that a small proportion of b3mem (about 5 - 10 yo of total) was functionally expressed at the cell surface, and that this anion-transport activity could be measured in intact yeast cells by a simple stilbene disulphonate-inhibitable chloride-uptake assay. By a series of expression time-course experiments, we demonstrated that functional b3mem was added to and removed from the plasma membrane at comparable rates (approximately 5-20 molecules/cell per min) provided that galactose was not limiting. We concluded that the biosynthesis, translocation to the plasma membrane, recycling and degradation of the b3mem polypeptide occurred with a half-life of approximately 1-3 h, and that the observed cell-surface b3mem was a dynamic steady-state population. In more recent studies, we have found that intact human band 3 (residues 1-911), the Nterminally truncated kidney isoform of human band 3 (residues 66-911) and b3mem (residues 361-91 1) were expressed at comparable levels. However, intact and kidney band 3 could not be detected in the plasma membrane using whole-cell uptake assays or immuno-electron microscopy (J. D. Groves, P. Falson, M . Thines, M. le Maire and M . J. A. Tanner, unpublished work). This observation is consistent with the results of Sekler et al. [31], and suggests that a portion of the band 3 cytoplasmic domain (possibly between residues 182 and 360) modifies the intracellular targeting pathway of the expressed protein. Cell-surface expression of b3mem in yeast provides a straightforward system to study the functional properties of band 3 by site-directed mutagenesis. However, samples of b3mem ran as multiple bands on Western immunoblots [32], indicating that the material produced under these conditions was not likely to be suitable for overexpression and purification for structural studies. Using a panel of monoclonal antibodies against b3mem, we confirmed that this lack of homogeneity was not due to N-glycosylation, but resulted from proteolytic cleavage in the third extracellular loop (generating 18- and 35-kDa fragments) and carboxypeptidase Y digestion of the C-terminus. By contrast, intact band 3 and kidney band 3 ran as single unglycosylated bands on immunoblots (J. D. Groves, P. Falson, M. le Maire and M . J. A. Tanner, unpublished work). It is likely that the greater level of post-translational processing of b3mem is due to the observed differences in intracellular targeting of the two polypeptides.

0 1999 Biochemical Society

Expression of band 3 in insect and mammalian cells Human band 3 has been expressed at the cell surface of recombinant baculovirus-infected insect cells, where functionality was confirmed by

920


T h e kidney isoform of mouse band 3 (residues 80-929) has been expressed in the intercalated kidney cell line IC250 [42]. T h e expressed protein was detected in the plasma membrane of transiently transfected cells using a short immunological tag which had been inserted into an exofacial loop position in the band 3 cDNA sequence. Beckmann and co-workers [43] have recently developed a novel heterologous expression system for human band 3 in the human K562 erythroleukaemia cell line. K562 cells do not express endogenous band 3 or a number of red-cell-surface antigens, but do contain GPA and various other erythroid proteins. Stable transfection of the K562 cell line is therefore a particularly suitable system with which to study the biosynthesis and interactions of band 3 with other red-cell proteins. These workers reported functional cell-surface expression of band 3 in K562 cells, and quantified the yield by stilbene disulphonate-sensitive chloride-efflux assays and Western immunoblotting. T h e estimated density of band 3 in the plasma membrane of K562 cells (350 moleculeslpm') was markedly higher than that reported to date in any other heterologous system (Table 1). This indicates the potential importance of other red-cell membrane proteins such as GPA and the Rhesus (Rh) polypeptides in high-level expression of band 3, and supports the view that many of these proteins may form a complex within the native erythrocyte membrane environment. Four isoforms of chicken band 3 have been found which differ in their N-terminal sequences. Transfection studies in human erythroleukaemia (HEL) cells have shown that the two most extensively truncated variants are targeted to the plasma membrane, whereas the longer forms are retained in intracellular membranes [44]. These results suggest that the N-terminal cytoplasmic sequence of chicken band 3 may act as a signal to direct the polypeptide to a particular intracellular compartment. A similar effect of N-terminal sequences was discussed above for intact band 3 or b3mem in the yeast expression system. Further work is required to determine whether such phenomena are of biological significance when observed in heterologous systems.

stilbene disulphonate-sensitive chloride uptake and efflux assays [33]. As in the yeast system, the expressed band 3 was not glycosylated, and was shown to possess similar kinetic properties to band 3 in red cells. There have been several reports of the expression of band 3 in mammalian cell lines. Ruetz and co-workers [34] expressed mouse band 3 by transient transfection of a human embryonic kidney cell line (HEK293) and showed that the expressed protein was retained in intracellular membranes. Crude microsomes were prepared from the transfected cells, reconstituted into proteoliposomes and the band 3 was confirmed to be functionally active [35]. However, a subsequent report indicates that the mouse band 3 cDNA used in these studies may have contained two spontaneous point mutations which caused intracellular retention without loss of functionality [21]. Timmer and co-workers [36,37] have recently reported stable expression of human band 3 in a clonal cell line derived from HEK293. Both Nglycosylated and unglycosylated forms of band 3 were expressed in the cells under the tight control of an ecdysone-inducible promoter. Immunofluorescence revealed intense staining of band 3 in the plasma membrane and perinuclear region and diffuse staining throughout the cytoplasm. Stilbene disulphonate-sensitive chloride-efflux assays were used to show that some expressed band 3 was functional in the plasma membrane, with the same kinetic characteristics as band 3 in red cells. T h e HEK293 system has been used to study the biosynthesis and inter-molecular associations of band 3 in a mammalian cell-membrane environment. Gomez and Morgans [38] examined the interaction of band 3 with the red-cell cytoskeletal protein ankyrin in HEK293 cells. They showed that newly synthesized band 3 binds ankyrin in the endoplasmic reticulum and may facilitate the exit of band 3 from this compartment, but not its movement to the cell surface. Purified microsomes from transfected HEK293 cells were used in an in vitro assay to show that ankyrin binds to mouse erythroid band 3 with much higher affinity than to the mouse kidney band 3 isoform, indicating an essential [39] though not sufficient [40] role for residues 1-79 in this interaction. T h e transmembrane topology of the anion-transport domain has been evaluated by glycosylationinsertion mutagenesis and expression in HEK293 cells [41] and shown to be similar to that observed previously in microsomal membranes using an in vitro system [S].

Conclusion Human red-cell band 3 has now been functionally expressed in all the principal eukaryotic heterologous expression systems. I n most instances a significant minority of the expressed polypeptides

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Biochemical Society Transactions ( 1999) Volume 27, part 6

could be detected at the cell surface using aniontransport assays. However, the yield of band 3 appears to be generally lower than that of some other model multispanning membrane proteins. For example, expression of Glut 1 has been estimated to be 7 ng/oocyte [45], i.e. 10" copies/cell, which is about 100-fold higher than for band 3, and sufficient quantity to allow detection by immunoblotting. T h e reported density of expressed band 3 (or b3mem) in the plasma membranes of three diverse cell types (oocytes, insect cells and yeast) were remarkably similar, although about 100-fold lower than that of band 3 in the red-cell membrane (Table 1). T h e highest density of cellsurface expression was reported in a mammalian cell line (K562) although this was still 20-fold lower than in the red-cell membrane. In the future, either Saccharomyces cerevisiae or Pichia pastoris may offer a cheap and efficient yeast over-expression system for milligram quantities of recombinant band 3, but at present the protocols require further development to reduce heterogeneity and increase yield. As our understanding of the key factors affecting the quality and quantity of expressed protein (i.e. biosynthesis, intracellular targeting, post-translational modifications, protein stability and host-cell viability) advances, we may start to be able to match the many parameters of the expression system to the particular application which it serves.

I 3 Karbach, D., Staub. M., Wood, P. G. and Passow, H. ( I 998) Biochim. Biophys. Acta 1371, I 14- I22 I 4 Tanner, M. J. A.. Martin. P. G. and High, S. ( I 988) Biochern. J. 256,703-7 I 2 I5 Garcia, A. M. and Lodish, H. F. ( I 989) J. Biol. Chern. 264, 19607-19613 I 6 Groves, J. D. and Tanner, M. J. A. ( I 992) J. Biol. Chern. 267, 22163-22170 I 7 Groves, J. D. and Tanner, M. 1. A. ( 1994) J, Membr. Biol. 140, 8 1-88 I 8 Groves, J. D. and Tanner, M. J. A. ( I 994) Mol. Membr. Biol. I I, 31-38 19 Chemova, M. N., Jarolirn, P., Palek, J. and Alper, S. L. (1995) J. Membr. Biol. 148, 203-2 I0 20 Tanner, M. J. A., Bruce, L J. and Groves, J. D.( I 997) in Membrane Proteins: Structure, Function and Expression Control (Harnasaki, N. and Mihara, K., eds.), pp, 353-372, Karger, Basel 21 Chemova, M. N., Humphries. B. D.. Robinson, D. H., StuartTilley, A. K., Garcia, A. M., Brosius, F. C. and Alper, S. L. ( 1997) Biochirn. Biophys. Acta 1329, I I I - I23 22 Groves, J. D. and Tanner, M. J. A. ( I 995) J. Biol. Chern. 270, 9097-9 I05 23 Wang, L., Groves, J. D.. Mawby, W. J. and Tanner, M. J. A. ( I 997) J. Biol. Chern. 272, I063 I -I 0638 24 Groves, J. D., Wang, L. and Tanner, M. J. A. ( I 998) Biochem. J. 332, 161-171 25 Groves, J. D., Wang. L. and Tanner, M. J. A. ( I 998) FEBS Lett. 433,223-227 26 Brosius, F. C., Alper, S. L., Garcia, A. M. and Lodish, H. F. ( I 989) J. Biol. Chern. 264, 7784-7787 27 Bruce, L. J., Cope, D. L., Jones,G. K., Schofield, A. E., Buriey, M., Povey, S., Unwin. R I., Wrong, 0.and Tanner, M. J. A. ( I 998) J. Clin. Invest. 100, I 693-1 707 28 Groves, J. D., Ring, S. M., Schofield, A. E. and Tanner, M. J. A. ( I 993) FEBS Lett. 330, 186-1 90 29 Fievet, B., Gabillat, N., Borgese, F. and Motais. R ( I 995) EMBO J. 14, 5 158-5 I 69 30 Grisshammer, R and Tate, C. G. ( I 995) Q. Rev. Biophys. 28, 3 I51122 3 I Sekler, I., Kopito, R and Casey, J. R ( I 995) J. Biol. Chem. 270,2 1028-2 I034 32 Groves, J. D., Falson. P., le Maire, M. and Tanner, M. J. A. ( 1996) Proc. Natl. Acad. Sci. U.S. A. 93, 12245- I2250 33 Dale, W. E., Textor, J. A., Mercer, R W. and Sirnchowitz, L ( 1996) Protein Expression Purif 7, I - I I 34 Ruetz, S., Lindsey, A. E., Ward, C. L. and Kopito, R. R ( I 993) J. Cell Biol. I 2 I, 37118 35 Sekler, I., Lo, R S., Mastrocola. T. and Kopito. R R ( I 995) J. Biol. Chern. 270, I I25 I- I I256 36 Timmer, R T., Saxena, N. C., Stockman, S., Yang. Y., Smith, P. M. and Gunn, R 6. ( 1998) J. Gen. Physiol. I 12, PI27 (abstract) 37 Timmer, R T. and Gunn, R B. (I 999) Am. J. Physiol. 276, C6K75 38 Gornez, S. and Morgans, C. ( I 993) J. Biol. Chern. 268, 19593-19597 39 Ding, Y., Casey, J. R and Kopito. R R (1 994) J. Biol. Chern. 269, 3220 1-32208 40 Ding, Y., Kobayashi, S. and Kopito, R R ( I 996) J. Biol. Chern. 27 I, 22494-22498 4 I Popov. M., Li, J.and Reithrneier, R A. F. (I 999) Biochem. J. 339.269-279

This work was supported by the Wellcorne Trust and an EMBO Short Term Fellowship. M.D.P. was the recipient of a BBSRC studentship. I Steck T. L. ( 1974) J. Cell Biol. 62, I I 9 2 Jennings,M. L. ( I 989) Annu. Rev. Biophys. Biophys. Chem. 18, 3971130 3 Reithrneier, R A. F. ( I 993) Curr. Opin. Struct. Biol. 3, 5 15-523 4 Tanner, M. J. A. ( 1997) Mol. Membr. Biol. 14, 155- I 65 5 Popov, M., Tarn, L. Y., Li, J. and Reithrneier, R A. F. ( I 997) I. Biol. Chern. 272, 18325- I8332 6 Wang, D. N.,Sarabia, V. E., Reithrneier, R A. F. and Kuhlbrandt, W. (I994) EMBO J. 13, 3230-3235 7 Wood, P. G. ( I 992) Progr. Cell Res. 2, 325-338 8 Morgan, M., Hanke. P., Grygorczyk R, Tintschl. A,, Fasold. H. and Passow. H. ( 1985) EMBO J. 4, 1927- I93 I 9 Kopito, R R and Lodish, H. F. ( I 985) Nature (London) 3 16, 234-238 10 Bartel, D., Lepke, S., Layh-Schrnitt. G., Legrurn. B. and P~SSOW, H. ( I 989) EMBO J. 8,360 1-3609 I I Lepke, S., Becker, A. and Passow, H. ( I 992) Biochirn. Biophys. A d a I 106, I 3- I 6. 12 Muller-Berger, S., Karbach, D.. Konig, J., Lepke. S.. Wood, P. G., Appelhans. H. and Passow. H. (I 995) Biochemistry 34,93 15-9324 -


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45 Gould, G. W. and Leinhardt, G. E. ( I 989) Biochemistry 28, 9447-9452

42 Glaser, M. J. and Edwards, J. C. (1994) J. Am. SOC. Nephrol. 5, P253 (abstract) 43 Beckrnann, R, Srnythe, J. S., Anstee, D. J. and Tanner, M. J. A. ( I 998) Blood 92,44284438 44 Cox, K. H.,Adair-Kik T. and Cox, J. V. ( I 995) J. Biol. Chem. 270, 19752-1 9760

Received I 8 June I999

Overexpression of eukaryotic membrane proteins in transgenic tobacco : pioneering the ‘green expression system ’ with the purification and crystallization of recombinant light-harvesting complex II Ralf Flachmann Botanisches Institut, Universitat Heidelberg, Irn Neuenheimer Feld 360, D-69 I 20 Heidelberg, Germany

Introduction

pure plant membrane proteins is still rare [13]. Most researchers make use of yeast for the overexpression of plant membrane proteins, e.g. for the triose phosphate translocator [14], for the monosaccharide-H+ symporter [15] or for the plasma membrane H+-ATPase [ 161. Although these proteins are purified from total yeast membranes in a single metal affinity chromatography step using recombinant histidine-tagged proteins [14,15], the lipid environment in which they are embedded is different and might lead to problems in some biochemical and structural studies.

T o date, detailed structures of only seven families of membrane proteins are known, and five of them are involved in photosynthesis and respiration. T h e three-dimensional structures of a few of these photosynthetic complexes and respiratory proteins have been determined : bacterial reaction centres [1,2], bacterial photosystem I [3,4] and bacterial cytochrome c oxidase [S], plant lightharvesting complex (LHCII) [6], and, at lower resolution, bacterial antenna complex (LH2) [7] and bacterial light-harvesting complex I (LHI) PI. These structural studies have been performed with naturally abundant proteins that can be purified in large quantities. Genetics and physiology in purple bacteria have allowed the introduction of mutations in reaction centres at residues that are involved in electron transfer and to accumulate sufficient quantities of these recombinant complexes. Several mutants have been crystallized and their structures resolved to around 3 A [9]. Homologous expression was one of the prerequisites which led to our detailed knowledge of reaction centres and their molecular function.

In vitro reconstitution versus in vivo expression Photosynthetic complexes like photosystem I1 and its antenna ( L H C I I ) depend on correctly folded protein structures that are formed and maintained by the interaction of all available pigment-binding sites with the protein moiety. Therefore, only organisms synthesizing both carotenoids and chlorophylls are able to accumulate functional complexes. When expressed in Escherichia coli, the D1 protein of photosystem I1 forms insoluble inclusion bodies and needs to be reconstituted in vitro with pigments to retain its native structure [17]. In vitro-reconstituted L H C I I has been found to form two-dimensional crystals with the same crystal symmetry as the native L H C I I [18]. However, both the relatively low resolution of 20-30 A with reconstituted L H C I I [18] and difficulties during reconstitution led to the setting up of an in planta expression system for several reasons. (i) In vitro reconstitution of the denatured L H C I I monomer with several pigments into functional trimers is cost-intensive as an excess of pure pigments is required and, dependent on the focus of the study, large amounts of detergents might become necessary. (ii) Due to the small

State of the art: overexpression of plant membrane proteins in yeast Homologous expression systems have been developed in bacteria, yeast, insect cells and mammalian cells [101. Although magnificent progress has been recently made with the structure determination of L H C I I [6] and photosystem I1 [11,121, homologous expression and isolation of

Abbreviations used: LHCII, plant light-harvesting complex; ER, endoplasrnic reticulum; NTA. nitrilotnacetic acid.

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