yeast Saccharomyces cerevisiae

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 12245-12250, October 1996 Biochemistry

Functional cell surface expression of the anion transport domain of human red cell band 3 (AE1) in the yeast Saccharomyces cerevisiae (heterologous expression/plasma membrane/chloride transport/membrane protein)

JONATHAN D. GROVES*t, PIERRE FALSONt, MARC LE MAIREt, AND MICHAEL J. A. TANNER* *Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 lTD, United Kingdom; and tSection de Biophysique des Proteines et des Membranes, Departement de Biologie Cellulaire et Moleculaire, Commissariat a l'Energie Atomique and Centre National de la Recherche Scientifique, Unite de Recherche Associee 2096, Commissariat a l'Energie Atomique de Saclay, F-91191 Gif-sur-Yvette, France

Communicated by Pierre Joliot, Institut de Biologie Physico-Chimique, Paris, France, August 14, 1996 (received for review May 28, 1996)

face of AE2-transfected cells (7). Chicken erythrocyte band 3 has been expressed in a human erythroleukemic cell line by transient transfection (8): whereas the complete chicken AE1 was found only in intracellular membranes, two naturally occurring variant forms with truncated N-termini were localized to the plasma membrane. Heterologous expression of mammalian red cell band 3 at the cell surface has to date been reported only in studies of cRNA injection ofXenopus oocytes (9-12), where the functional properties of both band 3 and B3mem were confirmed by anion transport assays. None of these expression systems has produced sufficiently large quantities of functional red cell band 3 for purification or structural analysis. Although yeast has been used extensively for heterologous gene expression (13, 14), the number of membrane proteins that have been functionally expressed is rather limited (reviewed in ref. 15). It would appear that mammalian membrane proteins are expressed in yeast less readily than plant or fungal proteins and in most cases accumulate in perinuclear membranes (predominantly the endoplasmic reticulum). A notable exception was the expression of the Na+/K+-ATPase (16), where ouabain-binding sites were detected in the plasma membrane of yeast. While the work described in this paper was in progress, the expression of a red cell band 3 recombinant (containing the first 10 amino acid residues of phosphoglycerate kinase, a six histidine residue affinity tag, and amino acid residues 183-911 of human AE1) was reported in intracellular membranes of yeast (17), using a constitutive promoter. In this paper, we have used the vector pYeDP1/8-10 (18), which has given functional overexpression of the sarcoplasmic reticulum Ca2+-ATPase (19) and of the CHIP 28 water channel (20). We report the establishment of a rapid galactose-inducible yeast expression system for the integral membrane anion transport domain of human red cell band 3. We show by a simple chloride influx assay that at least a proportion of the expressed protein is correctly folded in vivo and is targeted to the cell surface of the yeast cell, where it mediates stilbene disulfonate-sensitive anion transport.

We expressed the 52-kDa integral membrane ABSTRACT domain (B3mem) of the human erythrocyte anion transporter (band 3; AE1) in a protease-deficient strain of the yeast Saccharomyces cerevisiae under the control of the inducible GAL10-CYCl promoter. Immunoblots of total protein from transformed yeast cells confirmed that the B3mem polypeptide was overexpressed shortly after induction with galactose. Cell surface expression of the functional anion transporter was detected by using a simple transport assay to measure stilbene disulfonate-inhibitable chloride influx into intact yeast cells. The B3mem polypeptide was recycled and degraded by the cells with a half-life of approximately 1-3 hr, which led to a steady-state level of expression in exponentially growing cultures. Our data suggest that 5-10%o of total B3mem is functionally active at the cell surface at any one time and that overexpression of this anion transport protein does not interfere with cell growth or survival. This is one of only a few reports of the functional expression of a plasma membrane transport protein in the plasma membrane of yeast cells and to our knowledge is the first report of red cell band 3-mediated anion transport at the plasma membrane of cDNAtransformed cells. The cell surface expression system we describe will provide a simple means for future study of the functional properties of band 3 by using site-directed mutagenesis.

Band 3 (AE1) is the major integral membrane protein of the human red cell, being present at about 1.2 x 106 copies per cell (1), and facilitating the one-for-one exchange of Cl- and HCO3-. It has served as a model for investigating the structure and function of integral membrane proteins and has been studied intensively (reviewed in refs. 2-4). The protein comprises two distinct domains. The N-terminal 43-kDa cytoplasmic portion (amino acid residues 1-360) anchors band 3 to the red cell skeleton as well as certain glycolytic enzymes and hemoglobin. The C-terminal 52-kDa integral membrane domain (B3mem, amino acid residues 361-911) spans the red cell membrane up to 14 times and is both necessary and sufficient for the anion exchange function of the protein. Both red cell band 3 and the membrane domain portion exist almost exclusively as oligomers (reviewed in ref. 5), and a low-resolution three-dimensional crystal structure of dimeric band 3 has been

MATERIALS AND METHODS Plasmid Constructions. The cDNA encoding the membrane domain (amino acid residues 361-911) of human red cell band 3 (21) was inserted into the yeast expression vector pYeDP1/ 8-10 (pYeDP; ref. 18) under the control of the inducible GAL10-CYC1 hybrid promoter and the phosphoglycerate kinase terminator. The clone pBSXG1.b3 (11) acted as a template for the addition of linkers by polymerase chain reaction (PCR) amplification. The sense PCR primer was 5'-CGAAGCAATTGCCATGGGCCTAGACTTA-

published (6). Human and mouse erythrocyte band 3 have been expressed by transfection of human embryonic kidney cells but were not targeted to the cell surface and caused abnormal cell morphology (7). In contrast, the related mouse kidney anion transporter (AE2) was functionally expressed at the cell sur-

Abbreviations: B3mem, 52-kDa anion transport domain of human red cell band 3; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate; PNGase F, peptide-N-glycosidase F. tTo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 12245

12246

Biochemistry: Groves et al.

AATGG-3', which contains MfeI and NcoI restriction enzyme sites, an initiator methionine codon, and the 17-base nucleotide sequence which matches amino acids 361-366 of the band 3 cDNA. The antisense PCR primer was 5'-CGAAGCCGCGGTCACACAGGCATGGCCAC-3', which contains a SaclI restriction enzyme site, a terminator codon, and the 15-base nucleotide sequence which matches amino acids 911-907 of the band 3 cDNA. The PCR product was digested with MfeI and SaclI and ligated into EcoRI and SstII-digested vector, to yield pYeDP.b3mem. The entire coding sequence of pYeDP.b3mem was verified by DNA sequencing. Yeast Strains and Culture. Saccharomyces cerevisiae strain FKY282 (MATa, SRP40, pep4::LEU2, ura3-1, leu2-3,-112, his3-11,-15, trpl-1, ade2-1, kanamycin-resistant; supplied by F. Kepes, Commissariat a l'Energie Atomique de Saclay), which is protease-deficient, was used for transformation of pYeDPbased plasmids and cultured at 30°C. Untransformed yeast cells were grown on YPAD complete medium (22). Transformants were generated by electroporation (Bio-Rad gene pulser) and selected by ura3 complementation on plates of S6 glucose minimal medium (18). Cell density in liquid cultures was monitored by optical density at 600 nm (OD600); an OD600 of 1.0 corresponds to approximately 107 cells per ml (22). To induce expression, exponential-phase cultures in S6 medium were pelleted at 5000 x g for 5 min and resuspended directly into S5 galactose minimal selective medium (18). Anion Transport Assay. Cultures in S5 medium were washed by centrifugation at 5000 x g for 5 min, first in 0.5 vol of water and then in 10 ml of transport buffer (10 mM NaHepes, pH 7.4/50 mM NaCl). Cells were finally resuspended in transport buffer at an approximate density of OD600 = 100, which is about 15% (vol/vol) yeast cells. The exact cell density (within the range 80 < OD600 < 120) was measured in each case and results were normalized to OD600 = 100. To 20-,ul samples of cell suspension (containing about 2 x 107 cells), either 1 ,ul of water or 1 ,l of 2 mM 4,4'-diisothiocyanatostilbene-2,2'disulfonate (DIDS) was added to give a final concentration of 100 ,M in each tube. After 10 min at 18°C, the assay was started with 2 ,tl of Na36Cl solution (Amersham). This gave 60-70 mM NaCl at a specific activity of 5-6,umol of NaCl/,Ci and 10-12 ,uCi/ml (1 ,uCi = 37 kBq). The influx of 36C1- was usually measured after 90 s at 18°C with triplicate samples. Yeast cells were washed rapidly by twice adding 0.9 ml of wash buffer (10 mM NaHepes, pH 7.4/50 mM sodium gluconate/25 ,uM DIDS) and centrifuging at 10,000 x g for 10 s, and then radioactivity was measured. Yeast Cell Lysis and Immunoblotting. Yeast cultures were prepared as for the transport assay. Samples containing exactly 10 OD600 units of cells (about 108 cells) were combined with 400 ,ul of 2% (wt/vol) trichloroacetic acid (TCA). Cells were disrupted by vigorous Vortex mixing for 4 min with an equal volume of glass beads (0.5 mm diameter). The lysate was washed three times with 400 Al of TCA and the collections were pooled. After 15 min on ice, the precipitated proteins were pelleted by centrifugation at 13,000 x g for 10 min at 4°C. Samples were solubilized in SDS/PAGE buffer (100 ,tl) (23), separated on 10.5% acrylamide gels (10 ,u per sample), and immunoblotted (24). The antibodies used were BRIC170 (from D. Anstee, International Blood Group Reference Laboratory, Bristol, United Kingdom), BRIC132 (24), and BRIC155 (24). Rabbit polyclonal antiserum pHB3-4 raised against a peptide corresponding to amino acid residues 548565 of band 3 was also used (from K. Ridgwell, University of Bristol). Blots were visualized by using the ECL (enhanced chemiluminescence) method (Amersham) and Bio-Max MR film (Kodak). Quantification was performed by comparison of expressed protein with known numbers of human red blood cells (run on the same blot) using IMAGEMASTER scanning software (Pharmacia LKB). A conversion factor of 1.2 x 106 band 3 molecules per red cell was assumed (1).

Proc. Natl. Acad. Sci. USA 93 (1996)

Deglycosylation. Yeast cell protein was precipitated with trichloroacetic acid and then solubilized in 100 ,ul of buffer [0.0625% SDS/1.25% (wt/vol) octaethylene glycol monododecyl ether (C12E8), 125 mM Tris HCl, pH 8.0/6.25 mM EDTA/4 mM phenylmethylsulfonyl fluoride (PMSF)/100 ,ug of leupeptin per ml/50 ,tg of antipain per ml]. Samples were divided into two and treated. either with 40 units of peptideN-glycosidase F (PNGase F; Oxford Glycosystems, Oxford, U.K.) or with an equal volume (10 ,ul) of water. After incubation at 37°C for 16 hr, 35 ,ul of 4% (wt/vol) SDS/30% (wt/vol) glycerol/5% (wt/vol) 2-mercaptoethanol/1 mM PMSF was added and samples (30 ptl) were analyzed by SDS/PAGE and Western blotting. As a positive control for deglycosylation, cells transformed with pYeDP were combined with 0.5 Al of human red cells and treated similarly. RESULTS Functional Expression of B3mem in Yeast. We introduced the cDNA clone for the 52-kDa C-terminal membrane domain of band 3 (amino acids 361-911 of band 3; B3mem) into the yeast-bacterial shuttle vector pYeDP. This construct (pYeDP.B3mem) places the expression of the B3mem polypeptide in yeast under the control of the inducible GAL10CYCI hybrid promoter. To facilitate expression, the ATG initiator codon of the B3mem cDNA was located 2 bases downstream of the EcoRI site in the polylinker of the vector. The protease-deficient yeast strain FKY282 was transformed either with pYeDP.B3mem (B3mem cells) or with pYeDP vector (Y cells). B3mem cells and Y cells were grown first in S6 selective medium, which contains glucose, and then protein expression was induced in S5 selective medium, which contains galactose. After 14 hr of growth in S5 medium, the uptake of 36C1- into the cells was measured in the presence and absence of the membrane impermeant band 3 inhibitor DIDS (Fig. 1). The DIDS-sensitive chloride influx provides an estimate of the band 3-specific anion transport into the yeast cells. In the absence of DIDS, the mean 36C1- influx into B3mem cells was 20- to 30-fold higher than the influx into Y cells at each time point. In the presence of 100 ,uM DIDS, the 36CL- influx into B3mem cells was greatly reduced (80-95% inhibited) and was only slightly greater than that observed with Y cells. Subse-

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FIG. 1. Time course of chloride influx into yeast cells. B3mem cells (squares) or Y cells (circles) were grown in S5 medium for 14 hr at 30°C. Chloride influx (0-30 min) was measured in the absence (solid lines) or presence (broken lines) of 100 ,uM DIDS. Error bars represent the SEM of triplicate measurements. (Inset) Data for the initial 5 min, plotted with an expanded x-axis and contracted y-axis.


Biochemistry: Groves et al. quently 100 ,uM DIDS was used, as treatment

,uM DIDS gave similar results (data not shown). In the absence 36C1- influx into B3mem cells 500

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influx was followed by a slower results from the equilibration of other intracellular compartments. measured transport over a 90-s period, estimates the initial rate of chloride 1 Inset). Since an influx of 1 nmol Clcorresponds to about 0.1 fmol of Cl1 indicate that the DIDS-sensitive cells is about 0.17 fmol per cell (over an average rate of about 106 Cl- ions The expression of the B3mem polypeptide SDS/PAGE and immunoblotting three monoclonal antibodies: BRIC170, epitope at the N terminus (Fig. 2, directed against an intracellular cytoplasmic terminus (Fig. 2, lanes 3 and 4); and the C terminus (identical results to

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No bands were visualized with samples three antibodies detected a polypeptide mately 45 kDa with samples derived polypeptide, which represented the vealed by shorter film exposures narrowly separated doublet with tensely stained. Additional smaller samples from B3mem cells at approximate of 18 kDa (BRIC 170) or 35 kDa The apparent molecular masses of they were the N- and C-terminal cleavage in the predicted third extracellular tains the chymotrypsin site of band (25). This identification was confirmed with a polyclonal antibody (pHB3-4) from

intact

(data

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corresponding to amino which recognized intact B3mem but ments (data not shown). The quantity B3mem cells was estimated by using comparison with known amounts of sequence

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blood cells. With each of the three of intact B3mem polypeptide was pmol/OD unit of cells (i.e., about Making the assumption that the with the same efficiency as the intact 18-kDa product was estimated to of cells, which corresponds to approximately the expressed protein (lane 1); BRIC

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FIG. 2. Immunoblotting of B3mem (lanes 1 and 3) or Y cells (lanes 2 and 4) were incubated in 85 medium for 10 hr to final cell densities of 0.37 and 0.40 OD unit/ml, respectively. After SDS/PAGE and Western blotting (1 OD unit of cells per lane), the B3mem polypeptide was detected with BRIC170 (lanes 1 and 2) and BRIC132 (lanes 3 and 4). The 36C1- influx (90 expressed

s)

the

and absence of 100 ,uM DIDS B3mem, 0.12 and 1.64 nmol/OD unit of cells and nmol/OD unit of cells. in

presence

was as follows:

for Y, 0.10 and

35-kDa fragment was lower (0.05-0.06 pmol/OD unit of cells; lane 3), suggesting that this fragment may be degraded more readily than the 18-kDa product. Red cell band 3 is N-glycosylated at a single site, on Asn-642. To examine whether the B3mem polypeptide is N-glycosylated in yeast, B3mem cells were grow.n inS5 medium for 10.5 hr and 16.5 hr and proteins were treated with PNGase F. On BRIC132 immunoblots of PNGase F-treated and untreated samples, the apparent molecular masses of B3mem and the 35-kDa fragment were identical (data not shown). In contrast, a positive control sample of Y cells mixed with human red cells (containing red cell band 3) showed the expected reduction in molecular mass and tightening of the band in the presence of PNGase (data not shown). This was similar to the effect of PNGase on red cells alone and characteristic of deglycosylation of band 3. This result indicates that there is little or no N-glycosylation of B3mem in yeast cells expressing a high level of DIDS-sensitive chloride transport activity and concurs with previous findings thatN-glycosylation is not essential for band 3 or B3mem functionality (26, 27). Time Course of B3mem Expression. The induction of B3mem by galactose was followed in exponentially growing cells over a 14-hr period. The DIDS-sensitive chloride influx, cell growth, and quantity of expressed protein were measured every 2 hr (Fig. 3). After a short lag in cell growth as the culture adapted from glucose to galactose metabolism (0-4 hr), the culture entered an exponential growth phase (4-10 hr). Over this period the average doubling time of B3mem cells was approximately 4.7 hr, which was only slightly slower than the 4.3-hr doubling time of Y cells. Subsequently (10-14 hr), both B3mem and Y cells entered a more rapid phase of exponential cell growth with average doubling times of 1.7 and 1.5 hr, respectively. Since the growth rate of B3mem cells was only slightly slower than that of Y cells throughout, we conclude that the expression of functional B3mem does not interfere with the healthy growth of these cells. DIDS-sensitive chloride transport (Fig. 3A) was initially detectable at a very low level after 6 hr in S5 medium. Subsequently (6-10 hr), the DIDSsensitive chloride influx increased rapidly (at an average rate of 0.62 nmol/OD unit of cells per hr) to a maximum rate of transport (2.7 nmol of Cl- ions per OD unit of cells) after 10 hr S5 in medium. In contrast, Y cells expressed no DIDSsensitive chloride influx at any time. The induction of B3mem expression was monitored by immunoblotting with BRIC170 and BRIC132 (Fig. 3B). Results with BRIC155 (data not shown) were similar to those with BRIC132. The quantity of expressed B3mem closely reflected the amount of DIDSsensitive chloride transport at each time point, with the main increase occurring between 4 and 8 hr. This indicates that the time lag between biosynthesis and functional expression at the cell surface is relatively short (less than 1 hr) and comparable with the time taken for band 3 to reach the cell surface in erythroid precursor cells (28). During the period of maximum increase (between 6 and 8 hr) the average rate of B3mem biosynthesis was about 0.15 pmol/OD unit of cells per hr, which corresponds to about 150 molecules of B3memmin per per cell. Both the 18- and 35-kDa fragments were first detected after 8 hr in medium, and at each time a greater level of 18-kDa fragment was observed than of the 35-kDa fragment (as in Fig. 2). The proportion of the expressed protein that was present as 18-kDa fragment increased steadily throughout the experiment, consistent with the hypothesis that the 18-kDa fragment is more stable in the cells than the 35-kDa portion. Factors Affecting B3mem Expression. We examined the effect of longer induction times (>14 hr in S5 medium) on the yield, proteolysis, and cell surface expression of B3mem. In the firet experiment (Fig. A), for various times (14-21 hr) and DIDS-sensitive chloride influx and protein expression were estimated. After >15.5 hr, both B3mem and Y cell cultures attained stationary phase and

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12248

Proc. Natl. Acad. Sci. USA 93 (1996) A

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FIG. 3. Time course of B3mem expression. B3mem cells (squares) Y cells (circles) were cultured in S5 medium for various times from 4 to 14 hr. Flasks were inoculated with cells at densities in the range 0.05-0.4 OD unit of cells per ml to give exponentially growing cultures with final cell densities in the range 0.4-0.9 OD unit/ml. (A) DIDSsensitive chloride influx (solid lines) was determined from the difference between the mean influx (90 s) at each time in the presence and absence of 100 ,uM DIDS, from two replicate experiments each performed in triplicate. Relative cell growth (broken lines) is calculated from final density/initial density. (B) Intact B3mem together with the 18- and 35-kDa fragments were immunoblotted and scanned. The amount of expressed protein is shown. Cross-hatched bars, B3mem; solid bars, 18-kDa (BRIC170) or 35-kDa (BRIC132) fragor

ment.

B3mem cells had a reduced level of anion transport. Immunoblotting with BRIC 170 indicated that the yield of B3mem decreased steadily with time. However, the amount of 18-kDa fragment remained roughly constant between 15.5 and 21 hr and hence increased as a proportion of the total expressed protein as the culture aged. There was no DIDS-sensitive chloride transport activity in Y cells at any time (Fig. 4A) or in B3mem cells cultured only in S6 (data not shown). To investigate whether this decrease in chloride transport was caused by the length of time the B3mem was expressed in the cells or by exhaustion of the growth medium at high cell density, B3mem cells were incubated for 19.5 hr in S5 medium from three different initial densities (Fig. 4B). The culture containing the lowest cell density (0.01 OD unit/ml) remained in exponential growth phase throughout and exhibited relatively high levels of chloride transport and B3mem expression. In contrast, the cultures that were inoculated at the interme-

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FIG. 4. Effect of time and initial cell density on B3mem expression. (A) (Left) B3mem cells (squares) or Y cells (circles) were cultured in S5 medium for various times from 14 to 21 hr. DIDS-sensitive chloride influx (solid lines) was determined from two replicate experiments (B3mem) or one experiment (Y), each in triplicate. Cell growth was recorded as final cell density (broken lines) from an initial density of 0.05 OD unit/ml. (Right) Immunoblots of expressed protein (BRIC170) were scanned and quantified as in Fig. 3. Cross-hatched bars, B3mem; solid bars, 18-kDa fragment. (B) B3mem cells were inoculated into S5 medium at three different cell densities and incubated for 19.5 hr. DIDS-sensitive chloride influx (solid line), final cell density (broken line), and B3mem expression (bars) were determined as described for A.

diate (0.05 OD unit/ml) and higher (0.2 OD unit/ml) densities at various stages of the stationary phase and showed much lower levels of anion transport. In the case of the 0.2 OD unit/ml of culture, where the cells had been at stationary phase for about 6 hr before harvest, the yield of B3mem was particularly low. To confirm that prolonged expression of B3mem does not adversely affect growth or survival, B3mem cells were cultured in S5 medium for 17 hr, then diluted again into fresh medium for a second 17 hr period. In this case, the growth rates during the two incubations were comparable, and the DIDS-sensitive chloride influx after 34-hr expression was similar to that after 17 hr (data not shown). Taken together, the results in Fig. 4 indicate that it is essential to use actively growing cultures (OD600 < 1.5) to obtain high expression of B3mem (particularly for functional expression at the plasma membrane) and to minimize proteolysis. Biosynthesis, Cell Surface Expression, and Degradation of B3mem. In Fig. 3, a period of rapid induction of B3mem (4-10 hr) resulted in the expression of a high level of DIDS-sensitive chloride transport. Subsequently (10-14 hr), B3mem expression appeared to increase much more slowly and the DIDSsensitive chloride transport activity was slightly reduced. The following experiments were devised to investigate whether the biosynthesis of B3mem ceases after about 10 hr of expression

were



or whether biosynthesis and translocation to the plasma membrane continue but the onset of degradation prevents further accumulation of B3mem in the yeast cells. To determine the rate of translocation of B3mem to the surface of cells that are already expressing a high level of anion transport activity, a pulse-chase experiment was performed (Fig. 5). B3mem cells were incubated in S5 medium for 10.5 hr and then DIDS was added to the culture for a further 30 min. We assumed that DIDS would be able to bind covalently to yeast cell surface B3mem as it does to red cell band 3 during this time, leading to permanent inactivation of these molecules. Chloride transport activity and protein expression were measured before and after the DIDS pulse, and after a further 1-hr and 4-hr growth in the absence of DIDS. The pre-existing DIDS-sensitive chloride influx (Fig. SA, no. 1) was almost abolished by DIDS treatment (no. 2) but rapidly recovered during the chase period (nos. 3 and 4). About 60% of the initial transport activity was restored after 1 hr of further growth. However, immunoblots using either BRIC132 (Fig. SB) or BRIC170 (data not shown) showed that the total amount of B3mem expressed in the cells was not affected by the DIDS treatment. We conclude that molecules of B3mem continue to be translocated to the plasma membrane in cells that have attained an apparent steady-state level of expression. To investigate the rate of degradation of B3mem in yeast, B3mem cells were cultured in S5 medium for 10 hr and then transferred either to fresh S5 medium or to S5 medium containing cycloheximide at 100 ,ug/ml to inhibit de novo protein synthesis. DIDS-sensitive chloride influx, cell growth, and B3mem expression were measured after 1, 3, and 6 hr of further incubation (Fig. 6). The cells in S5 medium continued to grow in exponential phase until at least the 3-hr time point, resulting in high levels of DIDS-sensitive chloride influx at the 1- and 3-hr points and high levels of B3mem polypeptide throughout the experiment. In contrast, cell growth was arrested rapidly in the cycloheximide-treated cells, for which the rate of chloride transport and total B3mem content of the cells as measured by using BRIC170 (Fig. 6B) and BRIC155 (data A

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FIG. 6. Inhibition of B3mem biosynthesis with cycloheximide. B3mem cells were inoculated into S5 medium at 0.1 OD unit/ml and incubated for 10 hr to a final density of 0.6 OD unit/ml. Cells were centrifuged and resuspended in S5 medium at 0.4 OD unit/ml in the presence (triangles or +Cyc) or absence (squares or -Cyc) of cycloheximide at 100 ,ug/ml. Samples were taken after 1, 3, and 6 hr of incubation. (A) DIDS-sensitive chloride influx (solid lines) was determined from two replicate experiments each performed in triplicate. Cell growth is recorded as final cell density (broken lines). (B) Immunoblots of expressed protein (BRIC170) were scanned and quantified as in Fig. 3. Cross-hatched bars, B3mem; solid bars, 18-kDa fragment.

not shown) declined steadily with time. After 3 hr of incubation with cycloheximide, the quantity of B3mem polypeptide in these cells was only about 30% of that in untreated cells. This corresponds to an average rate of degradation of about 100 molecules of B3mem per cell per min. The 18- and 35-kDa fragments were degraded more slowly than the intact B3mem, which suggests that the cleavage to give these products may occur relatively late in the life-span of the B3mem molecule. The effects of cycloheximide are similar to those of high cell density (Fig. 4).

DISCUSSION

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FIG. 5. Inactivation of cell surface B3mem with DIDS. B3mem cells inoculated into S5 medium at 0.15 OD unit/ml and incubated for 10.5 hr to a cell density of 0.35 OD unit/ml. The culture was divided: one portion was prepared for anion transport and immunoblotting (no. 1); the remainder was incubated with DIDS (500 ,uM) for a further 30 min at 30°C, then cooled on ice andwashed three times at 4°Cwith ice-cold water (twice with 250 ml then once with 80 ml). The washed cells were divided: one portion (30 ml) was prepared as above (no. 2); the other portion (50 ml) was resuspended in S5 medium without DIDS at a cell density of 0.18 OD unit/ml. Samples were removed after a further 1-hr (no. 3, 0.2 OD unit/ml final density) and 4-hr (no. 4, 0.4 OD unit/ml final density) incubation and prepared as above. (A) DIDS-sensitive chloride influx, each point derived from the mean of two replicate experiments performed in triplicate. (B) Immunoblots of expressed protein (BRIC132) were scanned and quantified as in Fig. 3. Cross-hatched bars, B3mem; solid bars, 35-kDa fragment. were

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In this paper, we have demonstrated that transformed yeast cells can mediate a rapid biosynthesis of the anion transport domain of human erythrocyte band 3 (B3mem) and are able to target at least a proportion of the expressed protein in a correctly folded form to the cell surface. The levels of total cellular B3mem and DIDS-sensitive anion transport increase rapidly on induction with galactose, but subsequently both the total cellular B3mem and the functional polypeptide at the cell surface are turned over with a half-life of about 1-3 hr. Actively growing cells are able to maintain a steady-state level of B3mem. If biosynthesis is reduced, either by shortage of metabolites on aging of the culture or by specific inhibition, the levels of expressed B3mem decrease rapidly. High expression in yeast of a band 3 recombinant has been reported recently (17) for a constitutive expression system. This band 3 fusion protein was partially purified from yeast cells and after reconstitution into liposomes was shown to possess anion transport properties similar to those of red cell band 3. However, in this case the protein apparently was not targeted to the plasma membrane and expression interfered considerably with

growth. The turnover number of band 3 for chloride in human red cells has been reported to be approximately 5 x 104 ions per s at 38°C and 145 mM NaCl (29). Several factors would markedly reduce this value under the conditions of the yeast assay: first, the temperature of the assay (18°C), since Qlo = 3 in the range 15-38°C (29); second, the NaCl concentration (68 mM), which would give about 50% saturation (29); third, the yeast plasma membrane has a lipid composition different from that of the native red cell membrane; and fourth, the

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biosynthesis of B3mem in yeast occurs without the other red cell proteins with which it may normally interact, notably glycophorin A. Human red cells that naturally lack this protein (MkMk cells) show band 3-mediated transport that is about 60% of that in normal red cells, which is probably due to a subtle difference in the folded structure of the protein (30). Combining these factors, we can approximate that the chloride turnover of B3mem in yeast cells is likely to be about 1/50 of that of band 3 in the red cell assay-i.e., about 103 ions per s. From the data in Fig. 2, we can estimate the proportion of expressed B3mem that is located in the plasma membrane. The total quantity of B3mem is 0.3-0.4 pmol/OD unit of cells (about 2 x 104 molecules per cell); if this were all functionally active at the cell surface it would mediate an influx of about 2 x 107 Cl- ions per cell per s. The observed DIDS-sensitive influx was 1.52 nmol/OD unit of cells (about 106 Cl- ions per cell per s), which implies that about 5% of the total B3mem is expressed in an active form in the plasma membrane. Since the true initial rate of chloride transport may be slightly higher than the average influx over the first 90 s that we have measured (see Fig. 1 Inset), we conclude that about 5-10% of B3mem (i.e., 1-2 x 103 molecules per cell) is functionally expressed at the cell surface. Preliminary immunoelectron microscopy studies confirm that some B3mem is present in the plasma membrane (D. Thines-Sempoux, J.D.G., P.F., M.l.M., and M.J.A.T., unpublished results). Haploid yeast has a cell volume of 70 ,um3 (22), which corresponds to 0.7 ,ul/OD unit of cells. If equilibrium is achieved with a net influx of 4 nmol of Cl- per OD unit of cells (Fig. 1) and assuming 10% of cell volume is accessible to the chloride (the remainder being mostly cell wall and vacuoles), then the intracellular Cl- concentration would be at 60 mM, which is comparable to the extracellular concentration used in the assay. From the cell volume, we can estimate the surface area of the cell to be 80 ,um2 (ignoring infolding of the plasma membrane). The expression of 1-2 x 103 B3mem molecules per cell in the plasma membrane will correspond to a density of about 20 molecules per .tkm2. This is similar to the density of mouse band 3 expressed at the cell surface of oocytes (20-40 molecules per tum2; ref. 9) but rather lower than that of band 3 in red cells (7000 molecules per ,um2). An initial rate of influx of 106 Cl- ions per cell per s corresponds to a flux of approximately 20 fmol of Cl- per mm2 per s. The maximum rate of biosynthesis of B3mem (6-8 hr) was about 150 molecules per cell per min (Fig. 3). Over the same period, the average increase in DIDS-sensitive chloride influx was about 0.7 nmol/OD unit of cells per hr. Assuming the turnover number of B3mem in yeast calculated above, the average rate of translocation of B3mem to the cell surface would be about 5-10 molecules per cell per min, which is about 5% of total B3mem biosynthesis. The DIDS pulse-chase (Fig. 5) and cycloheximide (Fig. 6) experiments gave similar estimates of the rates of increase (10-20 B3mem molecules per cell per min) and decrease (5-10 B3mem molecules per cell per min) of functional B3mem to/from the cell surface. The transit time of functional B3mem at the plasma membrane (1-2 hr) is therefore similar to the half-life of the total population of B3mem molecules in the yeast cell. Oligomerization is a concentration-dependent process that is important in the export of many membrane proteins from the endoplasmic reticulum (31). Higher levels of expression of band 3 or B3mem increased the proportion translocated to the cell surface of Xenopus oocytes (9, 12). The large quantities of B3mem expressed in yeast cells in our galactose-inducible system may drive oligomerization and favor the translocation process. It is possible that the proportion of B3mem at the cell surface is rate limited by a step in the secretory pathway, such as exit from the Golgi complex. Excess B3mem may then be


targeted to one or more other intracellular compartments, where post-translational modifications (reviewed in ref. 13) may increase heterogeneity. For example, we have detected B3mem as a tightly spaced doublet on immunoblots and have shown that at least a proportion of B3mem molecules undergo a specific proteolytic cleavage. This proteolysis may help to prevent any deleterious effect that overexpression of B3mem might cause. While the yeast expression system we describe can yield 100 ,ug/liter at high cell density, our results suggest that exponentially growing cultures at slightly lower densities will give more homogeneous samples of biologically active protein for purification. In conclusion, we have shown that the anion transport domain of human red cell band 3 is translocated to the plasma membrane of yeast cells, where it can mediate chloride transport. The availability of this cell surface expression system will provide a simple means for the future study of the functional properties of band 3 by using site-directed mutagenesis. We thank Dr. F. Kepes for supplying yeast and Dr. D. Anstee and Dr. K. Ridgwell for antibodies. This work was supported by a European Molecular Biology Organization Short Term Fellowship to J.D.G. and by grants from the Wellcome Trust, the Commissariat a l'Energie Atomique, the Centre National de la Recherche Scientifique, and the Association Frangaise Contre les Myopathies. 1. Steck, T. L. (1978) J. Supramolec. Struct. 8, 311-324. 2. Jennings, M. L. (1989) Annu. Rev. Biophys. Biophys. Chem. 18, 397-430. 3. Reithmeier, R. A. F. (1993) Curr. Opin. Struct. Biol. 3, 515-523. 4. Tanner, M. J. A. (1993) Semin. Hematol. 30, 34-57. 5. Casey, J. R. & Reithmeier, R. A. F. (1991) J. Bio. Chem. 266, 15726-15737. 6. Wang, D. N., Sarabia, V. E., Reithmeier, R. A. F. & Kuhlbrandt, W. (1994) EMBO. J. 13, 3230-3235. 7. Ruetz, S., Lindsey, A. E., Ward, C. L. & Kopito, R. R. (1993) J. Cell Biol. 121, 37-48. 8. Cox, K. H., Adair-Kirk, T. L. & Cox, J. V. (1995) J. Bio. Chem. 270, 19752-19760. 9. Bartel, D., Lepke, S., Layh-Schmitt, G., Legrum, B. & Passow, H. (1989) EMBO J. 8, 3601-3609. 10. Garcia, A. M. & Lodish, H. F. (1989)1J Bio. Chem. 264,19607-19613. 11. Groves, J. D. & Tanner, M. J. A. (1992) J. Biol. Chem. 267, 2216322170. 12. Groves, J. D. & Tanner, M. J. A. (1994) J. Membr. Biol. 140, 81-88. 13. Romanos, M. A., Scorer, C. A. & Clari, J. J. (1992) Yeast 8, 423-488. 14. Connerton, I. F. (1994) in Membrane Protein Expression Systems: A User's Guide, (Portland, London), pp. 177-217. 15. Grisshammer, R. & Tate, C. G. (1995) Q. Rev. Biophys. 28, 315-422. 16. Horowitz, B., Ealke, K. A., Scheiner-Bobis, G., Randolph, G. R., Chen, C. Y., Hitzeman, R. A. & Farley, R. A. (1990) J Biol. Chem. 265, 4189-4192. 17. Sekler, I., Kopito, R. R. & Casey, J. R. (1995) J Biol. Chem. 270, 21028-21034. 18. Pompon, D. (1988) Eur. J Biochem. 177, 285-293. 19. Centeno, F., Descamps, S., Lompre, A.-M., Anger, M., Moutin, M.-J., Dupont, Y., Palmgren, M. G.,.Villalba, J. M., M0ller, J. V., Falson, P. & le Maire, M. (1994) FEBS Lett. 354, 117-122. 20. Laize, V., Rousselet, G., Verbavatz, J.-M., Berthonaud, V., Gobin, R., Roudier, N., Abrami, L., Ripoche, P. & Tacnet, F. (1995) FEBS Lett. 373, 269-274. 21. Tanner, M. J. A., Martin, P. G. & High, S. (1988) Biochem. J. 256, 703-712. 22. Sherman, F. (1991) Methods Enzymol. 194, 3-21. 23. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 24. Wainwright, S. D, Tanner, M. J. A., Martin, G. E. M., Yendle, J. E. & Holmes, C.-(1989) Biochem. J 258, 211-220. 25. Jennings, M. L. & Adams, M. F. (1981) Biochemistry 20, 7118-7122. 26. Casey, J. R., Pirraglia, C. A. & Reithmeier, R. A. F. (1992) J. Biol. Chem. 267, 11940-11948. 27. Groves, J. D. & Tanner, M. J. A. (1994) Mol. Membr. Biol. 11, 31-38. 28. Braell, W. A. & Lodish, H. F. (1981)J. Biol. Chem. 256, 11337-11344. 29. Brahm, J. (1977) J. Gen. Physiol. 70, 283-306. 30. Bruce, L. J., Groves, J. D., Okubo, Y., Thilaganathan, B. & Tanner, M. J. A. (1994) Blood. 84, 916-922. 31. Hurtley, S. M. & Helenius, A. (1989)Annu. Rev. Cell Biol. 5,277-307.