Functional (AE1) Saccharomyces · ofleupeptin per ml/50,tg ofantipain per ml]. Sampleswere divided...

6
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) ABSTRACT We expressed the 52-kDa integral membrane 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 polypep- tide 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 in- terfere 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 cDNA- transformed 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 mu- tagenesis. 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 com- prises two distinct domains. The N-terminal 43-kDa cytoplas- mic 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 do- main (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 exclu- sively as oligomers (reviewed in ref. 5), and a low-resolution three-dimensional crystal structure of dimeric band 3 has been 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 mor- phology (7). In contrast, the related mouse kidney anion transporter (AE2) was functionally expressed at the cell sur- 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 local- ized 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 of Xenopus 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 accu- mulate in perinuclear membranes (predominantly the endo- plasmic 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. 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- Abbreviations: B3mem, 52-kDa anion transport domain of human red cell band 3; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate; PN- Gase F, peptide-N-glycosidase F. tTo whom reprint requests should be addressed. 12245 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. Downloaded by guest on May 13, 2020

Transcript of Functional (AE1) Saccharomyces · ofleupeptin per ml/50,tg ofantipain per ml]. Sampleswere divided...

Page 1: Functional (AE1) Saccharomyces · ofleupeptin per ml/50,tg ofantipain per ml]. Sampleswere divided into two and treated.either with 40 units ofpeptide-N-glycosidase F (PNGase F; Oxford

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

Functional cell surface expression of the anion transport domainof human red cell band 3 (AE1) in theyeast 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 etdes Membranes, Departement de Biologie Cellulaire et Moleculaire, Commissariat a l'Energie Atomique and Centre National de la Recherche Scientifique, Unitede 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)

ABSTRACT We expressed the 52-kDa integral membranedomain (B3mem) of the human erythrocyte anion transporter(band 3; AE1) in a protease-deficient strain of the yeastSaccharomyces cerevisiae under the control of the inducibleGAL10-CYCl promoter. Immunoblots of total protein fromtransformed yeast cells confirmed that the B3mem polypep-tide was overexpressed shortly after induction with galactose.Cell surface expression of the functional anion transporterwas detected by using a simple transport assay to measurestilbene disulfonate-inhibitable chloride influx into intactyeast cells. The B3mem polypeptide was recycled and degradedby the cells with a half-life of approximately 1-3 hr, which ledto a steady-state level of expression in exponentially growingcultures. Our data suggest that 5-10%o of total B3mem isfunctionally active at the cell surface at any one time and thatoverexpression of this anion transport protein does not in-terfere with cell growth or survival. This is one of only a fewreports of the functional expression of a plasma membranetransport protein in the plasma membrane of yeast cells andto our knowledge is the first report of red cell band 3-mediatedanion transport at the plasma membrane of cDNA-transformed cells. The cell surface expression system wedescribe will provide a simple means for future study of thefunctional properties of band 3 by using site-directed mu-tagenesis.

Band 3 (AE1) is the major integral membrane protein of thehuman red cell, being present at about 1.2 x 106 copies per cell(1), and facilitating the one-for-one exchange of Cl- andHCO3-. It has served as a model for investigating the structureand function of integral membrane proteins and has beenstudied intensively (reviewed in refs. 2-4). The protein com-prises two distinct domains. The N-terminal 43-kDa cytoplas-mic portion (amino acid residues 1-360) anchors band 3 to thered cell skeleton as well as certain glycolytic enzymes andhemoglobin. The C-terminal 52-kDa integral membrane do-main (B3mem, amino acid residues 361-911) spans the red cellmembrane up to 14 times and is both necessary and sufficientfor the anion exchange function of the protein. Both red cellband 3 and the membrane domain portion exist almost exclu-sively as oligomers (reviewed in ref. 5), and a low-resolutionthree-dimensional crystal structure of dimeric band 3 has beenpublished (6).Human and mouse erythrocyte band 3 have been expressed

by transfection of human embryonic kidney cells but were nottargeted to the cell surface and caused abnormal cell mor-phology (7). In contrast, the related mouse kidney aniontransporter (AE2) was functionally expressed at the cell sur-

face of AE2-transfected cells (7). Chicken erythrocyte band 3has been expressed in a human erythroleukemic cell line bytransient transfection (8): whereas the complete chicken AE1was found only in intracellular membranes, two naturallyoccurring variant forms with truncated N-termini were local-ized to the plasma membrane. Heterologous expression ofmammalian red cell band 3 at the cell surface has to date beenreported only in studies of cRNA injection ofXenopus oocytes(9-12), where the functional properties of both band 3 andB3mem were confirmed by anion transport assays.None of these expression systems has produced sufficiently

large quantities of functional red cell band 3 for purification orstructural analysis. Although yeast has been used extensivelyfor heterologous gene expression (13, 14), the number ofmembrane proteins that have been functionally expressed israther limited (reviewed in ref. 15). It would appear thatmammalian membrane proteins are expressed in yeast lessreadily than plant or fungal proteins and in most cases accu-mulate in perinuclear membranes (predominantly the endo-plasmic reticulum). A notable exception was the expression ofthe Na+/K+-ATPase (16), where ouabain-binding sites weredetected in the plasma membrane of yeast. While the workdescribed in this paper was in progress, the expression of a redcell band 3 recombinant (containing the first 10 amino acidresidues of phosphoglycerate kinase, a six histidine residueaffinity tag, and amino acid residues 183-911 of human AE1)was reported in intracellular membranes of yeast (17), using aconstitutive promoter. In this paper, we have used the vectorpYeDP1/8-10 (18), which has given functional overexpressionof the sarcoplasmic reticulum Ca2+-ATPase (19) and of theCHIP 28 water channel (20). We report the establishment ofa rapid galactose-inducible yeast expression system for theintegral membrane anion transport domain of human red cellband 3. We show by a simple chloride influx assay that at leasta proportion of the expressed protein is correctly folded in vivoand is targeted to the cell surface of the yeast cell, where itmediates stilbene disulfonate-sensitive anion transport.

MATERIALS AND METHODSPlasmid Constructions. The cDNA encoding the membrane

domain (amino acid residues 361-911) of human red cell band3 (21) was inserted into the yeast expression vector pYeDP1/8-10 (pYeDP; ref. 18) under the control of the inducibleGAL10-CYC1 hybrid promoter and the phosphoglyceratekinase terminator. The clone pBSXG1.b3 (11) acted as atemplate for the addition of linkers by polymerase chainreaction (PCR) amplification. The sense PCR primer was5'-CGAAGCAATTGCCATGGGCCTAGACTTA-

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

12245

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

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

AATGG-3', which contains MfeI and NcoI restriction enzymesites, an initiator methionine codon, and the 17-base nucleo-tide sequence which matches amino acids 361-366 of the band3 cDNA. The antisense PCR primer was 5'-CGAAGCCGCG-GTCACACAGGCATGGCCAC-3', which contains a SaclIrestriction enzyme site, a terminator codon, and the 15-basenucleotide sequence which matches amino acids 911-907 of theband 3 cDNA. The PCR product was digested with MfeIand SaclI and ligated into EcoRI and SstII-digested vector, toyield pYeDP.b3mem. The entire coding sequence ofpYeDP.b3mem was verified by DNA sequencing.

Yeast Strains and Culture. Saccharomyces cerevisiae strainFKY282 (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), whichis protease-deficient, was used for transformation of pYeDP-based plasmids and cultured at 30°C. Untransformed yeastcells were grown on YPAD complete medium (22). Transfor-mants were generated by electroporation (Bio-Rad genepulser) and selected by ura3 complementation on plates of S6glucose minimal medium (18). Cell density in liquid cultureswas monitored by optical density at 600 nm (OD600); an OD600of 1.0 corresponds to approximately 107 cells per ml (22). Toinduce expression, exponential-phase cultures in S6 mediumwere pelleted at 5000 x g for 5 min and resuspended directlyinto 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 waterand then in 10 ml of transport buffer (10 mM NaHepes, pH7.4/50 mM NaCl). Cells were finally resuspended in transportbuffer at an approximate density of OD600 = 100, which isabout 15% (vol/vol) yeast cells. The exact cell density (withinthe range 80 < OD600 < 120) was measured in each case andresults were normalized to OD600 = 100. To 20-,ul samples ofcell suspension (containing about 2 x 107 cells), either 1 ,ul ofwater or 1 ,l of 2 mM 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) was added to give a final concentration of100 ,M in each tube. After 10 min at 18°C, the assay wasstarted with 2 ,tl of Na36Cl solution (Amersham). This gave60-70 mM NaCl at a specific activity of 5-6,umol of NaCl/,Ciand 10-12 ,uCi/ml (1 ,uCi = 37 kBq). The influx of 36C1- wasusually measured after 90 s at 18°C with triplicate samples.Yeast cells were washed rapidly by twice adding 0.9 ml of washbuffer (10 mM NaHepes, pH 7.4/50 mM sodium gluconate/25,uM DIDS) and centrifuging at 10,000 x g for 10 s, and thenradioactivity was measured.

Yeast Cell Lysis and Immunoblotting. Yeast cultures wereprepared as for the transport assay. Samples containing exactly10 OD600 units of cells (about 108 cells) were combined with400 ,ul of 2% (wt/vol) trichloroacetic acid (TCA). Cells weredisrupted by vigorous Vortex mixing for 4 min with an equalvolume of glass beads (0.5 mm diameter). The lysate waswashed three times with 400 Al of TCA and the collectionswere pooled. After 15 min on ice, the precipitated proteinswere 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), andimmunoblotted (24). The antibodies used were BRIC170(from D. Anstee, International Blood Group Reference Lab-oratory, Bristol, United Kingdom), BRIC132 (24), andBRIC155 (24). Rabbit polyclonal antiserum pHB3-4 raisedagainst a peptide corresponding to amino acid residues 548-565 of band 3 was also used (from K. Ridgwell, University ofBristol). Blots were visualized by using the ECL (enhancedchemiluminescence) method (Amersham) and Bio-Max MRfilm (Kodak). Quantification was performed by comparison ofexpressed protein with known numbers of human red bloodcells (run on the same blot) using IMAGEMASTER scanningsoftware (Pharmacia LKB). A conversion factor of 1.2 x 106band 3 molecules per red cell was assumed (1).

Deglycosylation. Yeast cell protein was precipitated withtrichloroacetic acid and then solubilized in 100 ,ul of buffer[0.0625% SDS/1.25% (wt/vol) octaethylene glycol monodo-decyl ether (C12E8), 125 mM Tris HCl, pH 8.0/6.25 mMEDTA/4 mM phenylmethylsulfonyl fluoride (PMSF)/100 ,ugof leupeptin per ml/50 ,tg of antipain per ml]. Samples weredivided into two and treated. either with 40 units of peptide-N-glycosidase F (PNGase F; Oxford Glycosystems, Oxford,U.K.) or with an equal volume (10 ,ul) of water. Afterincubation at 37°C for 16 hr, 35 ,ul of 4% (wt/vol) SDS/30%(wt/vol) glycerol/5% (wt/vol) 2-mercaptoethanol/1 mMPMSF was added and samples (30 ptl) were analyzed bySDS/PAGE and Western blotting. As a positive control fordeglycosylation, cells transformed with pYeDP were combinedwith 0.5 Al of human red cells and treated similarly.

RESULTSFunctional Expression of B3mem in Yeast. We introduced

the cDNA clone for the 52-kDa C-terminal membrane domainof band 3 (amino acids 361-911 of band 3; B3mem) into theyeast-bacterial shuttle vector pYeDP. This construct(pYeDP.B3mem) places the expression of the B3mempolypeptide in yeast under the control of the inducible GAL10-CYCI hybrid promoter. To facilitate expression, the ATGinitiator codon of the B3mem cDNA was located 2 basesdownstream of the EcoRI site in the polylinker of the vector.The protease-deficient yeast strain FKY282 was transformedeither with pYeDP.B3mem (B3mem cells) or with pYeDPvector (Y cells).B3mem cells and Y cells were grown first in S6 selective

medium, which contains glucose, and then protein expressionwas induced in S5 selective medium, which contains galactose.After 14 hr of growth in S5 medium, the uptake of 36C1- intothe cells was measured in the presence and absence of themembrane impermeant band 3 inhibitor DIDS (Fig. 1). TheDIDS-sensitive chloride influx provides an estimate of theband 3-specific anion transport into the yeast cells. In theabsence of DIDS, the mean 36C1- influx into B3mem cells was20- to 30-fold higher than the influx into Y cells at each timepoint. In the presence of 100 ,uM DIDS, the 36CL- influx intoB3mem cells was greatly reduced (80-95% inhibited) and wasonly slightly greater than that observed with Y cells. Subse-

5-

03~~.

0 10 20 30E 1-x

05

------------.--0 ~0 10o 2 30

Influx time (mins)

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 at30°C. Chloride influx (0-30 min) was measured in the absence (solidlines) or presence (broken lines) of 100 ,uM DIDS. Error barsrepresent the SEM of triplicate measurements. (Inset) Data for theinitial 5 min, plotted with an expanded x-axis and contracted y-axis.

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Proc. Natl. Acad. Sci. USA 93 (1996) 12247

quently 100 ,uM DIDS was used, as treatment ,uM

500 ,uM DIDS gave similar results over a 30-min

(data not shown). In the absence of DIDS,

36C1- influx into B3mem cells was biphasic:

36C1- influx was followed by a slower uptake,

results from the equilibration of radiolabeled

other intracellular compartments. In all subsequent

measured transport over a 90-s period, which

estimates the initial rate of chloride influx

1 Inset). Since an influx of 1 nmol of Cl- per

corresponds to about 0.1 fmol of Cl- per cell,

1 indicate that the DIDS-sensitive chloride

cells is about 0.17 fmol per cell (over the 90-s

an average rate of about 106 Cl- ions per

The expression of the B3mem polypeptide

SDS/PAGE and immunoblotting of total

three monoclonal antibodies: BRIC170,

epitope at the N terminus (Fig. 2, lanes

directed against an intracellular cytoplasmic

terminus (Fig. 2, lanes 3and 4); and BRIC155,

the C terminus (identical results to BRIC132,

No bands were visualized with samples

three antibodies detected a polypeptide

mately 45 kDa with samples derived from

polypeptide, which represented the intact

vealed by shorter film exposures (data

narrowly separated doublet with the upper

tensely stained. Additional smaller bands

samples from B3mem cells at approximate

of 18 kDa (BRIC 170) or 35 kDa (BRIC

The apparent molecular masses of these

they were the N- and C-terminal products

cleavage in the predicted third extracellular

tains the chymotrypsin site of band 3 at amino

(25). This identification was confirmed

with a polyclonal antibody (pHB3-4) raised

sequence corresponding to amino acids 548-565

which recognized intact B3mem but neither

ments (data not shown). The quantity of

B3mem cells was estimated by using scanning

comparison with known amounts of band

blood cells. With each of the three BRIC

of intact B3mem polypeptide was estimated

pmol/OD unit of cells (i.e., about 20,000

Making the assumption that the two fragments

with the same efficiency as the intact protein,

18-kDa product was estimated to be about

of cells, which corresponds to approximately

the expressed protein (lane 1); in contrast,

kDa Lane 11213141

139-84-

41.7- B3mem- _UW -B3mem479---17 8ka-32.0-

--35kDa

17.9 l8kDa

FIG. 2. Immunoblotting of B3mem expressed

(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 s)

in the presence and absence of 100 ,uM DIDS was as follows: for

B3mem, 0.12 and 1.64 nmol/OD unit of cells and for Y, 0.10 and 0.09

nmol/OD unit of cells.

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-glycosylatedin yeast, B3mem cells were grow.n inS5 medium for 10.5 hr and16.5 hr and proteins were treated with PNGase F. OnBRIC132 immunoblots of PNGase F-treated and untreatedsamples, the apparent molecular masses of B3mem and the35-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 inmolecular mass and tightening of the band in the presence ofPNGase (data not shown). This was similar to the effect ofPNGase on red cells alone and characteristic of deglycosyla-tion of band 3. This result indicates that there is little or no

N-glycosylation of B3mem in yeast cells expressing a high levelof DIDS-sensitive chloride transport activity and concurs withprevious findings thatN-glycosylation is not essential for band3 or B3mem functionality (26, 27).Time Course of B3mem Expression. The induction of

B3mem by galactose was followed in exponentially growingcells over a 14-hr period. The DIDS-sensitive chloride influx,cell growth, and quantity of expressed protein were measuredevery 2 hr (Fig. 3). After a short lag in cell growth as the cultureadapted from glucose to galactose metabolism (0-4 hr), theculture entered an exponential growth phase (4-10 hr). Overthis period the average doubling time of B3mem cells was

approximately 4.7 hr, which was only slightly slower than the4.3-hr doubling time of Y cells. Subsequently (10-14 hr), bothB3mem and Y cells entered a more rapid phase of exponentialcell growth with average doubling times of 1.7 and 1.5 hr,respectively. Since the growth rate of B3mem cells was onlyslightly slower than that of Y cells throughout, we concludethat the expression of functional B3mem does not interferewith the healthy growth of these cells. DIDS-sensitive chloridetransport (Fig. 3A) was initially detectable at a very low levelafter 6 hr in S5 medium. Subsequently (6-10 hr), the DIDS-sensitive chloride influx increased rapidly (at an average rateof 0.62 nmol/OD unit of cells per hr) to a maximum rate oftransport (2.7 nmol of Cl- ions per OD unit of cells) after 10hr inS5 medium. In contrast, Y cells expressed no DIDS-sensitive chloride influx at any time. The induction of B3memexpression was monitored by immunoblotting with BRIC170and BRIC132 (Fig. 3B). Results with BRIC155 (data notshown) were similar to those with BRIC132. The quantity ofexpressed B3mem closely reflected the amount of DIDS-sensitive chloride transport at each time point, with the mainincrease occurring between 4 and 8 hr. This indicates that thetime lag between biosynthesis and functional expression at thecell surface is relatively short (less than 1 hr) and comparablewith the time taken for band 3 to reach the cell surface inerythroid precursor cells (28). During the period of maximumincrease (between 6 and 8 hr) the average rate of B3membiosynthesis was about 0.15 pmol/OD unit of cells per hr,which corresponds to about 150 molecules of B3mem perminper cell. Both the 18- and 35-kDa fragments were first detectedafter 8 hr inS5 medium, and at each time a greater level of18-kDa fragment was observed than of the 35-kDa fragment(as in Fig. 2). The proportion of the expressed protein that waspresent as 18-kDa fragment increased steadily throughout theexperiment, consistent with the hypothesis that the 18-kDafragment is more stable in the cells than the 35-kDa portion.

Factors Affecting B3mem Expression. We examined theeffect of longer induction times (>14 hr in S5 medium) on theyield, proteolysis, and cell surface expression of B3mem. In thefiret experiment(Fig.4 A),B3mem cells were incubated for

various times (14-21 hr) and DIDS-sensitive chloride influxand protein expression were estimated. After >15.5 hr, bothB3mem and Y cell cultures attained stationary phase and

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

A A

Time (hr)

t cD 0o cq lt

e (hr) BRIC 170

- -.wssR---

----

sXlI

* zEl | | e .t

3 ,n >Rg;2

I-,)

-_

CO

.

(U)

7a)coZ-'

V

0)

70

BRIC 132

FIG. 3. Time course of B3mem expression. B3mem cells (squares)or Y cells (circles) were cultured in S5 medium for various times from4 to 14 hr. Flasks were inoculated with cells at densities in the range0.05-0.4 OD unit of cells per ml to give exponentially growing cultureswith final cell densities in the range 0.4-0.9 OD unit/ml. (A) DIDS-sensitive chloride influx (solid lines) was determined from the differ-ence between the mean influx (90 s) at each time in the presence andabsence of 100 ,uM DIDS, from two replicate experiments eachperformed in triplicate. Relative cell growth (broken lines) is calcu-lated from final density/initial density. (B) Intact B3mem togetherwith 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) frag-ment.

B3mem cells had a reduced level of anion transport. Immu-noblotting with BRIC 170 indicated that the yield of B3memdecreased steadily with time. However, the amount of 18-kDafragment remained roughly constant between 15.5 and 21 hrand hence increased as a proportion of the total expressedprotein as the culture aged. There was no DIDS-sensitivechloride transport activity in Y cells at any time (Fig. 4A) orin 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 inthe cells or by exhaustion of the growth medium at high celldensity, B3mem cells were incubated for 19.5 hr in S5 mediumfrom three different initial densities (Fig. 4B). The culturecontaining the lowest cell density (0.01 OD unit/ml) remainedin exponential growth phase throughout and exhibited rela-tively high levels of chloride transport and B3mem expression.In contrast, the cultures that were inoculated at the interme-

B

la

o 1.5a0

o I.EC

-- 0,5-

c0 IaC] Inil

*2!

.0e~0

*1 =8a

II 'O

0.01 0.05 0.2itial cell density (A600/mQ)

0.01 0.05 0.2Initial cell density (A60/mi)

FIG. 4. Effect of time and initial cell density on B3mem expression.(A) (Left) B3mem cells (squares) or Y cells (circles) were cultured inS5 medium for various times from 14 to 21 hr. DIDS-sensitive chlorideinflux (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 of0.05 OD unit/ml. (Right) Immunoblots of expressed protein(BRIC170) were scanned and quantified as in Fig. 3. Cross-hatchedbars, B3mem; solid bars, 18-kDa fragment. (B) B3mem cells wereinoculated into S5 medium at three different cell densities andincubated for 19.5 hr. DIDS-sensitive chloride influx (solid line), finalcell density (broken line), and B3mem expression (bars) were deter-mined as described for A.

diate (0.05 OD unit/ml) and higher (0.2 OD unit/ml) densitieswere at various stages of the stationary phase and showedmuch lower levels of anion transport. In the case of the 0.2 ODunit/ml of culture, where the cells had been at stationary phasefor about 6 hr before harvest, the yield of B3mem was

particularly low. To confirm that prolonged expression ofB3mem does not adversely affect growth or survival, B3memcells were cultured in S5 medium for 17 hr, then diluted againinto fresh medium for a second 17 hr period. In this case, thegrowth rates during the two incubations were comparable, andthe DIDS-sensitive chloride influx after 34-hr expression wassimilar to that after 17 hr (data not shown). Taken together,the results in Fig. 4 indicate that it is essential to use activelygrowing cultures (OD600 < 1.5) to obtain high expression ofB3mem (particularly for functional expression at the plasmamembrane) and to minimize proteolysis.

Biosynthesis, Cell Surface Expression, and Degradation ofB3mem. In Fig. 3, a period of rapid induction of B3mem (4-10hr) resulted in the expression of a high level of DIDS-sensitivechloride transport. Subsequently (10-14 hr), B3mem expres-sion appeared to increase much more slowly and the DIDS-sensitive chloride transport activity was slightly reduced. Thefollowing experiments were devised to investigate whether thebiosynthesis of B3mem ceases after about 10 hr of expression

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Proc. Natl. Acad. Sci. USA 93 (1996) 12249

or whether biosynthesis and translocation to the plasma mem-brane continue but the onset of degradation prevents furtheraccumulation 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 aniontransport activity, a pulse-chase experiment was performed(Fig. 5). B3mem cells were incubated in S5 medium for 10.5 hrand then DIDS was added to the culture for a further 30 min.We assumed that DIDS would be able to bind covalently toyeast cell surface B3mem as it does to red cell band 3 duringthis time, leading to permanent inactivation of these mole-cules. Chloride transport activity and protein expression weremeasured before and after the DIDS pulse, and after a further1-hr and 4-hr growth in the absence of DIDS. The pre-existingDIDS-sensitive chloride influx (Fig. SA, no. 1) was almostabolished by DIDS treatment (no. 2) but rapidly recoveredduring the chase period (nos. 3 and 4). About 60% of the initialtransport activity was restored after 1 hr of further growth.However, immunoblots using either BRIC132 (Fig. SB) orBRIC170 (data not shown) showed that the total amount ofB3mem expressed in the cells was not affected by the DIDStreatment. We conclude that molecules of B3mem continue tobe translocated to the plasma membrane in cells that haveattained 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 thentransferred either to fresh S5 medium or to S5 mediumcontaining cycloheximide at 100 ,ug/ml to inhibit de novoprotein synthesis. DIDS-sensitive chloride influx, cell growth,and B3mem expression were measured after 1, 3, and 6 hr offurther incubation (Fig. 6). The cells in S5 medium continuedto grow in exponential phase until at least the 3-hr time point,resulting in high levels of DIDS-sensitive chloride influx at the1- and 3-hr points and high levels of B3mem polypeptidethroughout the experiment. In contrast, cell growth was ar-rested rapidly in the cycloheximide-treated cells, for which therate of chloride transport and total B3mem content of the cellsas measured by using BRIC170 (Fig. 6B) and BRIC155 (data

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FIG. 5. Inactivation of cell surface B3mem with DIDS. B3mem cellswere inoculated into S5 medium at 0.15 OD unit/ml and incubated for10.5 hr to a cell density of 0.35 OD unit/ml. The culture was divided: oneportion was prepared for anion transport and immunoblotting (no. 1); theremainder was incubated with DIDS (500 ,uM) for a further 30 min at30°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 (50ml) was resuspended in S5 medium without DIDS at a cell density of 0.18OD unit/ml. Samples were removed after a further 1-hr (no. 3, 0.2 ODunit/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 per-formed 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.

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FIG. 6. Inhibition ofB3mem biosynthesis with cycloheximide. B3memcells were inoculated into S5 medium at 0.1 OD unit/ml and incubatedfor 10 hr to a final density of 0.6 OD unit/ml. Cells were centrifuged andresuspended in S5 medium at 0.4 OD unit/ml in the presence (trianglesor +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-sensitivechloride influx (solid lines) was determined from two replicate experi-ments each performed in triplicate. Cell growth is recorded as final celldensity (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 incubationwith cycloheximide, the quantity of B3mem polypeptide inthese cells was only about 30% of that in untreated cells. Thiscorresponds to an average rate of degradation of about 100molecules of B3mem per cell per min. The 18- and 35-kDafragments were degraded more slowly than the intact B3mem,which suggests that the cleavage to give these products mayoccur relatively late in the life-span of the B3mem molecule.The effects of cycloheximide are similar to those of high celldensity (Fig. 4).

DISCUSSIONIn this paper, we have demonstrated that transformed yeastcells can mediate a rapid biosynthesis of the anion transportdomain of human erythrocyte band 3 (B3mem) and are ableto target at least a proportion of the expressed protein in acorrectly folded form to the cell surface. The levels of totalcellular B3mem and DIDS-sensitive anion transport increaserapidly on induction with galactose, but subsequently both thetotal cellular B3mem and the functional polypeptide at the cellsurface are turned over with a half-life of about 1-3 hr. Activelygrowing cells are able to maintain a steady-state level ofB3mem. If biosynthesis is reduced, either by shortage ofmetabolites on aging of the culture or by specific inhibition, thelevels of expressed B3mem decrease rapidly. High expressionin yeast of a band 3 recombinant has been reported recently(17) for a constitutive expression system. This band 3 fusionprotein was partially purified from yeast cells and after recon-stitution into liposomes was shown to possess anion transportproperties similar to those of red cell band 3. However, in thiscase the protein apparently was not targeted to the plasmamembrane and expression interfered considerably withgrowth.The turnover number of band 3 for chloride in human red

cells has been reported to be approximately 5 x 104 ions pers at 38°C and 145 mM NaCl (29). Several factors wouldmarkedly reduce this value under the conditions of the yeastassay: 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 differentfrom that of the native red cell membrane; and fourth, the

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biosynthesis of B3mem in yeast occurs without the other redcell proteins with which it may normally interact, notablyglycophorin A. Human red cells that naturally lack this protein(MkMk cells) show band 3-mediated transport that is about60% of that in normal red cells, which is probably due to asubtle difference in the folded structure of the protein (30).Combining these factors, we can approximate that the chlorideturnover of B3mem in yeast cells is likely to be about 1/50 ofthat 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 ofexpressed B3mem that is located in the plasma membrane. Thetotal quantity of B3mem is 0.3-0.4 pmol/OD unit of cells(about 2 x 104 molecules per cell); if this were all functionallyactive at the cell surface it would mediate an influx of about2 x 107 Cl- ions per cell per s. The observed DIDS-sensitiveinflux was 1.52 nmol/OD unit of cells (about 106 Cl- ions percell per s), which implies that about 5% of the total B3mem isexpressed in an active form in the plasma membrane. Since thetrue initial rate of chloride transport may be slightly higherthan the average influx over the first 90 s that we havemeasured (see Fig. 1 Inset), we conclude that about 5-10% ofB3mem (i.e., 1-2 x 103 molecules per cell) is functionallyexpressed at the cell surface. Preliminary immunoelectronmicroscopy studies confirm that some B3mem is present in theplasma 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 isachieved 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 thechloride (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 inthe assay. From the cell volume, we can estimate the surfacearea of the cell to be 80 ,um2 (ignoring infolding of the plasmamembrane). The expression of 1-2 x 103 B3mem moleculesper cell in the plasma membrane will correspond to a densityof about 20 molecules per .tkm2. This is similar to the densityofmouse band 3 expressed at the cell surface of oocytes (20-40molecules per tum2; ref. 9) but rather lower than that of band3 in red cells (7000 molecules per ,um2). An initial rate of influxof 106 Cl- ions per cell per s corresponds to a flux ofapproximately 20 fmol of Cl- per mm2 per s. The maximumrate of biosynthesis of B3mem (6-8 hr) was about 150 mole-cules per cell per min (Fig. 3). Over the same period, theaverage increase in DIDS-sensitive chloride influx was about0.7 nmol/OD unit of cells per hr. Assuming the turnovernumber of B3mem in yeast calculated above, the average rateof translocation of B3mem to the cell surface would be about5-10 molecules per cell per min, which is about 5% of totalB3mem biosynthesis. The DIDS pulse-chase (Fig. 5) andcycloheximide (Fig. 6) experiments gave similar estimates ofthe rates of increase (10-20 B3mem molecules per cell permin) and decrease (5-10 B3mem molecules per cell per min)of functional B3mem to/from the cell surface. The transit timeof functional B3mem at the plasma membrane (1-2 hr) istherefore similar to the half-life of the total population ofB3mem molecules in the yeast cell.

Oligomerization is a concentration-dependent process thatis important in the export of many membrane proteins fromthe endoplasmic reticulum (31). Higher levels of expression ofband 3 or B3mem increased the proportion translocated to thecell surface ofXenopus oocytes (9, 12). The large quantities ofB3mem expressed in yeast cells in our galactose-induciblesystem may drive oligomerization and favor the translocationprocess. It is possible that the proportion of B3mem at the cellsurface is rate limited by a step in the secretory pathway, suchas 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 detectedB3mem as a tightly spaced doublet on immunoblots and haveshown that at least a proportion of B3mem molecules undergoa specific proteolytic cleavage. This proteolysis may help toprevent any deleterious effect that overexpression of B3memmight cause. While the yeast expression system we describe canyield 100 ,ug/liter at high cell density, our results suggest thatexponentially growing cultures at slightly lower densities willgive more homogeneous samples of biologically active proteinfor purification.

In conclusion, we have shown that the anion transportdomain of human red cell band 3 is translocated to the plasmamembrane of yeast cells, where it can mediate chloride trans-port. The availability of this cell surface expression system willprovide a simple means for the future study of the functionalproperties 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 Euro-pean Molecular Biology Organization Short Term Fellowship toJ.D.G. and by grants from the Wellcome Trust, the Commissariat al'Energie Atomique, the Centre National de la Recherche Scienti-fique, 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,

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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,

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