Identification of revertants for the cystic fibrosis ΔF508 mutation using STE6-CFTR chimeras in yeast

Cell, Vol. 73, 335-346,

April 23, 1993, Copyright

0 1993 by Cell Press

Identification of Revertants for the Cystic Fibrosis AF508 Mutation Using STE6-CFTR Chimeras in Yeast John L. Teem,‘t Herbert A. Berger; Lynda S. Ostedgaard,’ Devra P. Rich,” Lap-Chee Tsui,t and Michael J. Welsh’ *Howard Hughes Medical Institute University of Iowa College of Medicine Departments of Internal Medicine and Physiology and Biophysics Iowa City, Iowa 52242 fDepartment of Genetics Hospital for Sick Children Toronto, Ontario M5G 1X8 Canada

Summary Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis; the most common mutation is deletion of phenylalanine at position 508 (AF508). We constructed STEG-CFTR chimeras with portions of the first nucleotide-binding domain (NBDl) of the yeast STE6 a-factor transporter replaced by portions of CFTR NBDl. The chimeras were functional in yeast, but mating efficiency decreased when AF508 was introduced into NBDl. We isolated two AF508 revertant mutations (R553M and R553Q) that restored mating; both were located within the CFTR NBDl sequence. Introduction of these revertant mutations into human CFTR partially corrected the processing and Cl- channel gating defects caused by the AF508 mutation. These results suggest that the NBDls of CFTR and STE6 share a similar structure and function and that, in CFTR, the regions containing F508 and R553 interact. They also indicate that the abnormal conformation produced by AF508 can be partially corrected by additional alterations in the protein. Introduction The cystic fibrosis transmembrane conductance regulator (CFTR) (Riordan et al., 1989; Rommens et al., 1989) is a member of a superfamily of structurally related membrane proteins named the traffic ATPases (Ames et al., 1990) or ATP-binding cassette (ABC) transporters (Hyde et al., 1990). The principal distinguishing feature of this superfamily is a highly conserved nucleotide-binding domain (NBD). Many members of the family transport small molecules across biological membranes in an ATP-dependent manner. However, CFTR is a Cl- channel regulated by phosphorylation and by cytosolic ATP (Anderson et al., 1991 b; Bear et al., 1992; Tabcharani et al., 1991; Berger et al., 1991; Cheng et al., 1991). The two NBDs play a central role in the function of CFTR Cl- channels; they interact directly with ATP to open the CFTR Cl- channel, and ATP hydrolysis may be required for this effect (Anderson et al., 1991a; Anderson and Welsh, 1992). The impor-

tance of the NBDs is further emphasized by the large number of cystic fibrosis (CF)-associated mutations that have been found in these domains (Kerem et al., 1990; Tsui, 1992). The most common CF-associated mutation, accounting for approximately 68% of CF chromosomes, is the deletion of phenylalanine at position 508 (AF508) in the middle of NBDl. CF is characterized by a loss of apical membrane CIchannel activity in epithelial cells (Knowles et al., 1983a, 1983b). The AF508 mutation causes loss of apical membrane Cl- channel activity by affecting the CFTR protein in two ways. First, the mutation causes defective processing and hence mislocalization of the mutant protein (Cheng et al., 1990; Denning et al., 1992c; Kartner et al., 1992). Abnormal localization of CFTRAF508 is presumed to result from misfolding of the mutant protein so that it fails to exit from the endoplasmic reticulum and progress to the Golgi complex and the apical membrane (Cheng et al., 1990). The Cl- transport defect associated with the AF508 mutation is thus largely a consequence of the absence of functional CFTR at the plasma membrane. Defective trafficking of CfTRAF508 can be partially reversed if cells are grown at lowered temperature. When CFTRA F508 is processed at a reduced temperature (less than 37%), some of the mutant protein transits the Golgi complex normally and is localized correctly in the plasma membrane (Denning et al., 1992a). Second, the AF508 mutation affects the function of the Cl- channel. Single-channel analysis of CFTRAF508 suggests that although the mutant protein is functional, it has an open state probability (PO) that is one-third that of wild-type CFTR (Denning et al., 1992a; Dalemans et al., 1991). Since CFTRAF508 retains partial function, a possible therapy for CF might involve pharmacologic intervention designed to correct the processing defect so that more CFTRAF508 reaches the plasma membrane. One way to understand the mechanism by which the AF508 mutation alters the localization and function of CFTR is to isolate revertant mutations in CFTRAF508 that compensate for these defects. By isolating AF508 revertant mutations that occur within the CFTR gene, one could determine the extent to which the CFTRAF508 CItransport defect can be corrected. Such an approach could also identify potential interactions of F508 with other amino acid residues in the protein and identify structural regions of CFTR that might be manipulated to restorefunction to CFTRAF508. Construction and identification of revertants of the CFTRAF508 Cl- transport defect in mammalian cells is impractical, since revertants cannot easily be selected based upon restoration of cAMPstimulated Cl- transport. As an alternative, we devised a method for the selection of AF508 revertants in yeast. To establish a phenotype for the AF508 mutation in yeast, we exploited the structural similarity between CFTR and another member of the traffic ATPase/ABC transporter family, the yeast STEG gene product. The STE6 gene of Saccharomyces CereViSiae en-

Cell 336

codes an integral membrane protein that functions to transport the peptide a-factor out of the cell during mating (Kuchler and Thorner, 1992; McGrath and Varshavsky, 1989). Deletion of the STEGgenefrom yeast (ste6d) results in a sterile phenotype. We constructed a STEG-CFTR hybrid gene containing approximately half of NBDl of CFTR, which complements a yeast ste6d mutation. Introduction of the AF508 mutation into the STEG-CFTR hybrid gene results in loss of a-factor transport, which is easily detected by a yeast cell mating assay. We then isolated second site mutations within the CFTR portion of the STEG-CFTR hybrid gene that revert the AF508 mating defect. These revertant mutations isolated in yeast were subsequently reconstructed into a CFTRAF508 cDNA gene and analyzed in mammalian cells. Results CFTR NBDl Sequences Can Replace STEG NBDl Sequences To assess the functional similarity of NBDl of CFTR and STEG, chimeric STf6 genes were constructed in which segments of the NBDl of STEG were substituted by the analogous sequences from CFTR. Chimeric gene constructs were transformed into a sfe6d yeast strain and tested by a quantitative cell mating assay to determine whether segments of CFTR could functionally replace the corresponding regions of STEG NBDl (Figure 1). A STEG-CFTR chimeric gene (Hl) was constructed with theDNAsequencefortheentireNBD1 ofSTE6(from N377 to A535) replaced by the corresponding region of CFTR (D443 to Y577) (Figure 1A). Yeast transformants containing the Hl plasmid were unable to complement the sre6d mutation (Figure 1B). Likewise, the STEG-CFTR chimera H2 that replaces the amino-terminal half of NBDl with CFTR sequences was also nonfunctional. These results suggest that the amino-terminal region of NBDl from CFTR cannot substitute for that of STEG. In contrast, STEG-CFTR chimeras containing the central part of NBDl from CFTR (H3 and H4) maintained a-factor transport activity comparable with the wild-type STEGyeast strain (Figure 1 B). This suggests that amino acid residues between the conserved Walker A and B motifs (Walker et al., 1982), although different in the NBDl of CFTR and STEG, provide a similar overall structure and function. When the chimeras included progressively larger segments of the carboxy-terminal region of NBDl from CFTR (H5 and H6), mating efficiency was reduced. Yeast transformed with the hybrid gene H5, which contained a substitution of 74 residues of STE6 NBDl (from R441 to 1518) by that from CFTR (from F494 to L558), mated at 12% the efficiency of wild-type STEG. Mating efficiency was further reduced in H6 to background levels, suggesting that the carboxy-terminal segment of STE6 NBDl is not interchangeable with that of CFTR. The AF508 Mutation Inhibits Function of a STEG-CFTR Hybrid A previous study suggested that missense mutations STEG that were analogous to CF-associated mutations

in in

NBDl result in defective a-factor transport (Berkower and Michaelis, 1991). However, single amino acid deletions analogous to AF508 within the central region of STE6 NBDl had no effect on STE6 function. Since F508 is clearly important for the function of CFTR, we hypothesized that this residue may also be important to the function of a STEG-CFTR hybrid transporter that contained CFTR sequences in NBDl. To assess the effect of the AF508 mutation on the function of STEG-CFTR hybrid transporters, the AF508 mutation was introduced into H3, H4, H5, and H6 (resulting in H3-AF508, H4-AF508, H5-AF508, and H6-AF508, respectively). Figure 1 B shows the effect of the AF508 mutation on a-factor transport as assessed by the quantitative yeast cell mating assay. Yeast transformed with the H3AF508 chimera mated with an efficiency equal to the H3 control (no AF508 mutation). A modest decrease in mating efficiency (40%) was observed for the strain containing the H4-AF508 chimera, relative to the H4 control. However, the AF508 mutation lowered mating efficiency by 80-fold in the H5-AF508 chimera as compared with H5. Thus, the AF508 mutation inhibits a-factor transport in STEG-CFTR hybrid transporters that contain a large replacement of STE6 NBDl with CFTR. The effect of the F508 deletion is most severe in the H5AF508 transporter, which contains the largest functional substitution of CFTR into STEG. This suggests that in the H5 chimera, NBDl residues from CFTR have a functional conformation similar to that in native CFTR and that deletion of F508 disturbs this conformation, resulting in decreased a-factor transport. In contrast, because the NBDl of the H3 and H4 transporters contain less CFTR sequence, the presence of F508 may not be critical to the folding or function of those chimeras. Revertants of AF508 Mating Defect in Yeast The difference between the H5 chimera (in which the AF508 mutation has a large effect) and the H4 chimera (in which the AF508 mutation has minimal effect) is six amino acids from CFTR: R553-L558. This region is of particular interest in CFTR and other trafficking ATPases, as it is directly adjacent to the conserved LSGGQ linker sequence of NBDl , postulated to function as a transducer of signals between the hydrophobic domains and the NBDs (Mimura et al., 1991). The R553-L558 region is also not present in the H3 chimera, in which the AF508 mutation has no effect. These observations suggest that there is a requirement for F508 (or amino acids adjacent to F508) in chimeras that also contain the amino acids R553-L558. Possibly there is an interaction between these two regions of the NBD, required for correct folding or function (or both) of the NBD. Deletion of F508 might disturb this intramolecular interaction, resulting in loss of function or mislocalization (or both) of the transporter. Thus, we speculated that a mutation in this region (R553-L558) might suppress the adverse effects of AF508 in NBDl. To isolate revertants of the H5-AF508 mating defect, the H5-AF508 construct was mutagenized in the R553L558 region in vitro by site-specific oligonucleotide mutagenesis (Ho et al., 1989). The mutagenesis method was

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(A) Amino acid sequence alignment of the NBDl of STE6 and CFTR. Identity between the two sequences is indicated by vertical bars. The arrow refers to the CFTR NBDI sequences that replace the corresponding region of STE6 in the H5 STEG-CFTR hybrid construct, The conserved Walker motifs are shown by the thick lines. (B) The NBDl of CFTR and STE6 aligned as in (A), with stippled bars denoting the extent of CFTR NBDl sequence replacing STE6 NBDl in plasmids encoding S7E6-CFTR hybrid genes Hl-H6. The portion of the CFTR amino acid sequence inserted into STE6 for the different hybrids is as follows: Hl, D443-Y577; H2, D443-F508; H3, F494-F508; H4, F494-S1546; H5, F494-L558; H6, F494-Y577. The mating efficiency of each strain containing a STEG-CFTR plasmid was determined by quantitative yeast mating assays, with the SEM as follows: Hl ,0.00019%; H2,0.0005%; H3, 10.1%; H3-AF508, 11.7%; H4, 9.4%; H4-AF508, 8.2%; H5, 3.2%; H5-AF508, 0.01%; H6, 0.00019%; H6-AF508, O.OtJO19%. Values are calculated from four separate experiments. The mating efficiency of the ste&l strain was determined to be 0.0005% of the wild-type STEG control. The position of the CFTR sequence RARISL is shown in the darkly stippled box.

designed to introduce random amino acid substitutions at single codon positions within the R553-L558 region of the H5-AF508 plasmid. The procedure should allow 20 possible substitutions at each amino acid position within R553-L558. The mutagenized DNA was transformed into yeast, and transformant colonies were analyzed by cell mating assays on petri dishes to identify colonies with a mating efficiency higher than the unmutagenized H5AF508 control. We identified two colonies with higher efficiencies of mating (Figure 2) within the first 20 mutant transformants analyzed. These yeast transformants each contained an H5-AF508 plasmid with a mutation at amino acid R553 of CFTR; in one case, R553 was replaced by

methionine (H5-AF508/R553M), and in the other plasmid, R553 was replaced by glutamine (H5-AF508IR553Q). It is possible that other mutations within the R553-L558 region of the H5-AF508 plasmid could also result in increased mating efficiency; however, we proceeded to analyze the R553Q and R553M mutants in greater detail without further mutagenesis of the R553-L558 region. The relative mating efficiency of the AF508 revertant strains are shown in Table 1, with results expressed relative to the original H5 strain. Whereas the mating efficiency of the H5-AF508 yeast strain is approximately lo/a of the H5 strain, yeast containing the H5-AF508/R553Q and H5AF508/R553M plasmids mated at 3% and 3204 respec-

Cell 330

Figure 2. Complementation CFTR Hybrid Genes

STE6 +

H5AF508

tively. The revertant mutations, therefore, partially suppress the AF508 mating defect. The R553M mutation alone had little effect on H5 (H5R553M); when this mutant was transformed into yeast, no further increase in mating efficiency was observed as compared with yeast containing H5 (Table 1). We speculate that defective a-factor transport by the H5-AF508 chimera occurs as a result of a change in the folding or function (or both) of the NBDl due to the AF508 mutation and that the AF508 mutation similarly affects the NBDI of CFTR. The R553Q and R553M mutations partially correct the defect in the H5-AF508 chimera and should also correct the defect in CFTRAF508 if a similar structure exists for NBDl in both proteins. As a test of this hypothesis, we introduced the R553Q and R553M mutations into CFTRAF508 cDNA and transfected mammalian cells with these constructs to determine whether the revertant mutations would correct the defect in CAMPregulated Cl- transport of CFTRAF508. R553Q and R553M Suppress the CFTRAF508 Anion Transport Defect We assessed the effect of the R553Q and R553M mutations on CFTR function by assaying for CAMP-stimulated halide efflux using the halide-sensitive fluorophore 8-methoxy-N-(3sulfopropyl)-quinolinium (SPQ) (Illsley and Verkman, 1987). Expression of CFTR cDNA containing either the R553Q or the R553M mutation alone (without the AF508 mutation) in HeLa cells generated CAMP-stimulated halide efflux like wild-type CFTR (Figure 3). As we previously reported (Rich et al., 1990), cells expressing the AF508 allele in this recombinant system showed little, if any, CAMP-stimulated halide efflux (Fig-

Table

1. Relative

Genotype H5R553M H5-AF508/R553M

H&AF508/R553Q HSAF508 Mating efficiencies and are expressed means f SEM.

Mating

Efficiency

of AF5OS Revertants

Mating Efficiency to H5 (%) 77.4

f

Relative

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0.5 by quantitative mating assays relative to H5 (100%). Data are

of sfe6d

by STEG-

Diploid colonies are shown resulting from cell matings between yeast strains JPYPOI (transformed with chimeras H5, H5AF508, H5AF5081R553Q and H5-AF5081R553M) and 22-2D. Colony density is proportional to the a-factor secretion by the STEG-CFTR hybrid transporter in each JPY201 transformant. Control strains wild-type STE6 and sfe6d consisted of JPY201 transformed with plasmids JTS6 (containing wild-type STEG) and JTSGT (containing the STE6 gene with the yeast TRW gene inserted within NSDI), respectively.

ure 3). However, when the mutations R553Q and R553M were introduced into CFTRAF508 (CFTRAF508/R553Q and CFTRAF508/R553M, respectively), CAMP-dependent anion permeability was restored. These results indicate that the CFTR Cl- channel defect observed with the AF508 mutant could be suppressed by either R553 mutation. As it is used in these experiments, the SPQ halide efflux provides only a qualitative assessment of the mutant CFTR channels. However, two observations about these data should be noted. First, the initial rate of halide efflux after CAMP stimulation was less for CFTRAF508IR553M and CFTRAF508/R553Q than for wild-type CFTR (Figure 3). Second, the number of cells responding to CAMP after transfection with the two revertant mutants was approximately 10% of the number of cells that responded after transfection with wild-type CFTR. This would be expected if the revertant mutants had only a low level of Cl- channel function so that only a subset of the transfected cells with the highest expression of CFTR were detected. Thus, CAMP-stimulated halide efflux from the CFTRAF5081 R553Q- and CFTRAF508/R553Mtransfected cells was less efficient than that in cells transfected with wild-type CFTR.ThisobservationsuggeststhattheAF508CI-channel defect was only partially reversed by the reversion mutations. Correction of CFTRAF508 Processing and Localization Cl- transport by CFTRAF508 containing the R553Q and R553M mutations would be detected only if the processing defect of CFTRAF508 was suppressed. To determine further whether the suppressor mutations correct the processing defect associated with the AF508 mutation, we examined the glycosylation patterns of CFTR and the various mutants expressed in HeLa cells (Figure 4). We have previously shown that CFTR is a glycoprotein that undergoes progressive glycosylation, resulting in three bands that migrate at different rates on an SDS-polyacrylamide gel (Gregory et al., 1990). Band A is the most rapidly migrating and represents the nascent, unglycosylated protein; band B has an intermediate rate of migration and a pattern of core glycosylation consistent with processing in the endoplasmic reticulum; band C migrates most slowly and has a pattern of mature glycosylation consistent with processing in the Golgi complex. CFTRAF508 is only present as the unglycosylated band A and the core glycosyl-

Revertants

339

of

CFTRAF508

6000

Figure 3. SPQ Halide Efflux Transfected HeLa Cells Expressing leles

-CFTR AF508/R553M -cR553M + AF508lR553Q I

R553Q

,+

AF508

Assay of CFTR Al-

NOa- was substituted for I- in the bathing medium at 0 min. Cells were stimulated 5 min later (arrow) with 20 uM forskolin and 100 uM isobutylmethylxanthine (CAMP). Data are presented as the fluorescence at time t (Ft) minus the baseline fluorescence (Fo; the average fluorescence measured in the presence of I-for 2 min prior to ion substitution). Data are mean ‘+ SEM and are representative of responses obtained in each condition. Experiments were performed in duplicate on each transfected culture, and at least three different transfected cultures were studied for each condition.

0

0

2

4

6

6

10

12

14

16

18

TIME (MIN)

ated band the Golgi (Cheng et We first HeLa cells

B protein, consistent with its failure to traverse complex and reach the plasma membrane al., 1990) (Figure 4). examined the glycosylation state of CFTR in expressing CFTR cDNA containing either the

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4. Analysis

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3

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(A) Expression of wild-type CFTR (lane I), CFlWR553M (lane 2) CFTR/R553Q (lane 3) and CFTRAF508 (lane 4) in HeLa cells. Mocktransfected HeLa cells are shown in lane 5. Cells were infected with recombinant vaccinia virus expressing T7 polymerase and transfected with plasmid DNA encoding CFTR expressed under the control of the T7 promoter. Lysates were prepared 8-10 hr posttransfection, and the immunoprecipitates prepared using antibody Ml-4 were labeled in vitro using PKA and [y-52P]ATP. The positions of bands A, B, and C are indicated. Autoradiography was for 24 hr. (B) Expression of wild-type CFTR (lane 1) or CFTR mutants CFTRAF508/R553M (lane 2) CFTRAF508IR553Q (lane 3) and CFTRAF508 (lane 4) in HeLa cells. Mock-infected HeLa cells are shown in lane 5. Cells were infected with two recombinant vaccinia viruses, one encoding CFTR variants and the other one encoding T7 polymerase. lmmunoprecipitates were prepared as in (A).

R553Q and R553M mutations alone (without the AF508 mutation). As shown in Figure 4A, band C is present in cells expressing wild-type CFTR (lane 1) and also mutant CFTR containing either the R553Q or R553M mutation (lanes 2 and 3). In contrast, only bands A and Bare present in cell8 expressing CFTRAF508 (lane 4). Thus, the R5530 and R553M mutationsalone do not affect the glycosylation of CFTR. In cells transfected with the CFTRAF508/R553M (Figure 48, lane 2) a small increase in band C is detectable as compared with CFTRAF508 (lane 4). This result was observed in three separate experiments. We were not able to consistently detect band C CFTR in cells expressing CFTRAF508/R553Q, possibly owing to limitation8 in the sensitivity of the assay. Thus, a detectable increase in band C occurs only with CFTRAF508/R553M as compared with CFTRAF508. However, the total amount of fully glycosylated CFTRAF508/R553M is still small relative to wild-type CFfR. To assess further the ability of suppressor mutations to correct the mislocalization defect of CFTRAF508, we used immunocytochemistry to detect CFTR at the cell surface in HeLa cells expressing wild-type or mutant CFfR. We used an antibody specific to an external epitope in the first extracellular loop of CFTR (Denning et al., 1992b). As we previously showed (Denning et al., 1992b), wild-type CFTR was detected at the surface of unpermeabilized HeLa cells (Figure 5A). In contrast, no CFTR was detectableatthesurfaceof cellsexpressingCFTRAF508(Figure 58). However, when cells expressed CFTRAF508/ R553M, CFTR was detected at the plasma membrane (Figure 5C). For CFTRAF508/R553Q, plasma membrane staining of CFTR was weak and variable and could not be demonstrated consistently (Figure 5D). Only nonspecific staining was Observed with preimmune serum or in the absence of CFTR (Figures 5E and 5F). These results are consistent with the observation that more CFTRAF5081 R553M than CFTRAF508Kt553Q is found in the band C

Figure

5. lmmunolocalization

HeLa cells were Cells were fixed and biotinylated transfected with

of Wild-Type

and Mutant

CFTR

transfected with wild-type CFTR (A) and CFTR mutants CFTRAF508 (B), CFTRAF508/R553M (C), and CFTRAF5081R553Q (D). (but not permeabilized) and stained using monoclonal antibody M84 (specific for an epitope in the first extracellular loop of CFTR) secondary mouse antibody. Fluorescence was detected from strepavidin-fluorescein isothiocyanate. Controls included HeLa cells wild-type CFTR and stained using preimmune antibody (E) and mock-infected cells stained using monoclonal antibody M84 (F).

form and suggest that the amount of CFTRAF508/R553Q at the plasma membrane is exceedingly low. Thus, these data indicate that only the CFTRAF508/R553M mutant is detectable in the plasma membrane at levels greater than that observed for CFTRAF508.

Single-Channel Analysis of CFTRAF508/R553Q CFTR To determine whether the suppressor mutations altered the single-channel properties of CFTRAF508, the revertant mutant CFTRAF508/R553Q was expressed in

Revertants 341

of CFTRAF508

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Figure 6. Single-Channel Current Activated by PKA and ATP in Excised, Inside-Out Patches from HeLa Cells Transiently Expressing CFTRAF50tllR553Cl or CFTRIR5530 The pipette solution contained 50 mM Cl-, and the bath solution contained 140 mM Cl-. (A) Tracings from a HeLa cell transiently expressing CFTWR553Q after addition of PKA (75 nM) and ATP (1 mM); no channel activity was observed prior to addition of both agents. Tracings were obtained at the indicated voltages. The broken line represents the closed state of the channel. (6) Single-channel current-voltage relation for CFTRIR5530. Each data point represents the mean f SEM of 4-12 experiments; in many cases the error bars are hidden by the symbol. (C) Tracings from a HeLa cell transiently expressing CFTRAF5OWR553Q after addition of PKA(75nM)andATP(l mM); nochannelactivity was observed prior to addition of both agents. (D) Single-channel current-voltage relation for CFTRAF5081R5530. Each data point represents the mean ? SEM of 4-7 experiments.

1P d1

HeLa cells and analyzed with the patch-clamp technique. We chose the R553Q mutant because, as discussed below, it has been observed in a CF patient. Single-channel analysis of CFTRAF508 indicates that the P, of the CFTRAF508 Cl- channel is reduced as compared with wild-type CFTR: Denning et al. (1992a) reported that CFTRAF508 had a P, of 0.13 f 0.01 (after incubation at reduced temperature), compared with a value of 0.34 + 0.02 for wild-type, and Dalemans et al. (1991) reported that the P, of CFTRAF508 Cl- channels ranged from 0.05 to 0.10, compared with a range of 0.22 to 0.35 for wild type. As expected from the response observed in the SPQ studies, CFTRIR553Q and CFTRAF5081R553Q formed functional Cl- channels (Figure 6). Inside-out membrane patches from either mutant showed no Cl- channel activity under basal conditions. However, addition of protein kinase A (PKA) and ATP to the cytosolic surface of the membrane patch activated channels in both cases; both agents were required to activate the channels (n = 6 for each mutant). Figures 6A and 6C show single-channel currents for CFTRIR553Q and CFTRAF508/R5530 Cl- channels after they had been activated by PKA and ATP. Singlechannel events were recorded from +60 mV to -120 mV in increments of 20 mV. The channels were Cl- selective

.4ms

as indicated by the reversal potentials (E,,, = 20 mV; EC, = 27 mV) (Figures 6B and 6D). CFTRIR553Q had a singlechannel slope conductance (from -120 mV to 0 mV) of 9.8 -c 0.5 pS (n = 7) and CFTRAF5081R553Q had a conductance of 10.2 f 0.2 pS (n = 11). The current measured at -100 mV for CFTRIR5530 (-1.1 & 0.1 pA, n = 6) and CFTRAF5081R553Q (-1.2 + 0.0 pA, n = 5) was not different from the current measured for wild-type CFTR (-1.1 f 0.0 pA, n = 7) (Anderson and Welsh, 1992). Thus, these data indicate that neither mutation altered the single-channel conductive properties of the CFTR CIchannel, nor did the mutations abolish the regulation by PKA. The results also indicate that, as predicted by the functional analysis of CFTRAF508/R553Q by the SPQ halide efflux assay above, at least some CFTRAF508/R5530 is localized in the plasma membrane. Following activation with PKA and ATP, we measured the P, for CFfRIR553Q and CFTRAF508/R553Q at different MgATP concentrations. Figures 7A and 78 show examples of the activity of single channels at different concentrations of MgATP; as the concentration of MgATP increased, both CFTRIR553Q and CFTRAF508/R553Q spent more time in the open state. Figure 7C shows that the P, of CFTRIR553Q Cl- channels was similar to that previously reported for wild-type CFTR Cl- channels (An-

Cdl 342

A

B AF508/R553Q

R553Q

Figure 7. The Effect of MgATP on Channel Activity in Excised, Inside-Out Membrane Patches from HeLa Cells Expressing CFTWR553Q and CFTRAF508IR553Q Following activation with PKA (75 nM) and ATP (1 mM), the P.of these mutants was measured at different MgATP concentrations. The broken line represents the closed state of the channel. The holding voltage was -60 mV. A single CFTWR553Q Cl- channel (A) and a single CFTFtAF506/R553Q Cl-channel (6) are shown at different MgATP concentrations. (C)The P, plotted against MgATP concentration for CFTRAF506/R5530 (n = 7) and for CFTR/ R553Cl (n = 6). Data for wild-type CFTR and CFTRAF506 (after incubation at reduced temperature) are from Anderson and Welsh (1992) and Denning et al. (1992a), respectively.

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MgATP

(mi)

derson and Welsh, 1992). However, at 1 mM ATP, the P, of CFTRAF5081R553Q (0.29 -c 0.02, n = 7) was greater than that of CFTRAF508 Cl- channels (0.13 + 0.01, n = 4) (Figure 7C). Thus, the R553Q mutation corrected the functional defect in gating of the CFTRAF508 Clchannels. Discussion Use of Hybrid Yeast Genes to Study Disease-Associated Mutations in Human Genes We have developed a novel approach that takes advantage of the power of yeast genetics to study a mutation that causes human disease. Using this approach, the most common CF mutation, AF508, can be assayed in yeast as a defect in cell mating. This approach avoids the inherent difficulty of using mammalian cells for genetic manipulations of CFTR based on functional assays. Genetic manipulation of the AF508 mutation in yeast allowed us to identify revertants that corrected the functional defect in the yeast model and also to show that the revertants partially corrected function of the human gene product. These results provide new insight into the effect of the AF508 mutation on the structure and function of CFTR. They also suggest a general method by which other human genetic disease mutations could be studied in yeast, provided a phenotype in yeast can be established.

Revertants of the AF508 Mutation Deletion of F508 produces two known defects in CFTR: it disrupts normal processing so that the protein is retained in the endoplasmic reticulum and fails to traffic to the plasma membrane (Cheng et al., 1990; Denning et al., 1992c; Kartner et al., 1992) and it alters channel gating so that the P, is reduced to values approximately one-third that of wild-type CFTR (Dalemans et al., 1991; Denning et al., 1992a). The revertants we have identified modify both defects. Mutation of R553 to methionine partially corrects the processing of CFTRAF508 as assessed by three criteria: it increased the amount of protein that was processed to the mature, fully glycosylated band C form; it increased the appearance of the mutant protein in the plasma membrane as determined immunocytochemically; and it produced functional channels in the plasma membrane when measured by the SPQ halide efflux assay. Although the R553Q revertant was less eff icient at correcting processing of CFTRAF508 as measured by the amount of fully processed CFTR present and by immunocytochemistry, it produced functional channels as measured by the SPQ halide efflux assay. In addition, R553Q corrected the altered gating of CFTRAF508. The effect of this mutation on channel function appeared to be greater than the effect on processing; the P, reverted to approximately that of wild-type CFTR, whereas processing remained much less than that of wild-type protein.

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of CFTRAF508

Previous data suggest that deletion of F508 causes an abnormal conformation or misfolding of the protein (Cheng et al., 1990). This notion is also consistent with the finding that the conformation of a peptide containing a part of NBDl from CFTR is altered by the AF508 mutation (Thomas et al., 1992). We speculate that the R553Q mutation must change the conformation of the CFTRAF5081 R553Q protein sufficiently to revert gating to normal and that both CFTRAF508lR553Q and CFTRAF508lR553M must adopt a conformation that allows at least some of the mutant protein to escape the cellular quality control mechanisms. However, since only a small amount of the revertant protein fully matures and proceeds to the cell surface, the conformation of NBDl must still remain distinctlydifferent from that of wild-type CFTR’in these recombinant cells. The CFTRAF508 revertant mutations R553Q and R553M were initially identified as revertants of the mating defect in yeast containing the H5-AF508 chimera, suggesting that the structure of NBDl in the chimera (and the effect of CF mutations on NBDl structure) resembles that of CFTR. Whether the H5-AF508 mating defect occurs as a result of misprocessing or misfunction (or both) of the H5-AF508 transporter remains to be determined and will require probes capable of precisely localizing the mutant protein in yeast. Our results suggest, however, that the yeast model system will provide a useful tool in further reversion analysis of CF-associated mutations of the NBDl of CFTR. It also may be useful in screening for pharmacologic compounds that correct the mating defect and could thus have therapeutic benefits for correction of the processing or functional defect of CFTRAF508. Implications for the Structure of CFTR NBDl There are several mechanisms by which these mutations might suppress the effects of a deletion of F508 in CFTR. The simplest interpretation is that amino acid R553 interacts directly with the central region of NBDl containing F508. Such an interaction might be essential for proper regulation of the channel, and it might contribute to the correct folding of CFTR. If AF508 disturbed such an interaction, the R553 suppressor mutations might act as compensatory mutations, restoring this interaction and allowing proper folding and normal function. Because wild-type phenotypes were observed with R553Q and R553M mutations alone, these mutations may be compensatory mutations that cause no detectable phenotype themselves. An alternative possibility is that deletion of F508 may generate a new interaction with the region around R553 that is detrimental to CFTR folding and function. For example, loss of F508 might displace the normal position of R553 and cause a misfunction and a misfolding of CFTR. Distinguishing between these and other alternatives will require further investigation of the structure and function of NBDl in CFTR. The sequence similarity of NBDl of CFTR to other traffic ATPases/ABC transporters (such as STEG) suggests that the NBDs share a common structure and function. This suggestion is supported by the fact that 50% of the NBDl of STE6 can be functionally replaced by the analogous

sequence from CFTR. Models of the NBDs from members of the traffic ATPase/ABC transporter superfamily have been proposed based upon their structural similarity to other ATP-binding proteins, such as adenylate kinase (Hyde et al., 1990; Ames et al., 1990). These models differ widely with respect to the proposed structure of the central region of the NBDl, as this region contains the greatest sequence divergence among members of the traffic ATPase/ABC transporter superfamily. The position of the F508 and R553 residues within NBDl cannot be unambiguously modeled based upon the similarity of CFTR to other ATP-binding proteins. Similarly, our genetic data cannot unambiguously define the positions of these residues within the tertiary structure of NBDl. However, our data indicate that these residues interact and may reside near to one another in NBDl. Implications for CF Could a AF508 revertant mutation correct the mislocalization and misfunction of the CFTRAF508 channel sufficiently so as to ameliorate the clinical manifestations of disease? Interestingly, a CF patient with both the AF508 and R553Q mutations on the same chromosome has been described (Dork et al., 1991). On the non-AF508 chromosome, the nonsense mutation R553X was found. Although this patient was reported to have severe disease (pancreatic insufficiency and lung disease), the sweat Cl- concentration for the patient (63 mmolll) was found to be close to the normal range of sweat Cl- concentration (O-60 mmolll, measured within the same clinic). In contrast, the mean value for patients (n = 80) homozygous for the AF508 mutation in that clinic was 109 mmolll and was 86 mmolll for patients (n = 9) heterozygous for R553X and AF508; these values are characteristic of CF patients and significantly higher than the normal range. It is interesting to speculate that the R553Q mutation may partially suppress the AF508 Cl- transport defect in the sweat gland in this patient, but is unable to suppress the defect in other affected tissues, such as the lung and pancreas, sufficiently to prevent clinical disease. Although the R553Q mutation corrected the defect in the P, of CFTRAF508, it was less effective than R553M in correcting the processing defect of CFTRAF508. Thus, the in vivo effect of the R553Q mutation on processing and function of CFTRAF508 might be quite small. However, a combination of restored CFTRAF508 channel function and improved processing might be sufficient to allow some of the CFTRAF508/R553Q mutant protein to reach the plasma membrane, where it could mediate Cl- transport in the duct of the sweat gland. In this regard, one might predict that the R553M mutation (or the AF508/R553Q on both chromosomes) might have a greater effect in suppressing the AF508 Cl- transport defect in the sweat gland and other organs. Experimental Procedures Chemicals and Solutions Catalytic subunit of CAMP-dependent protein kinase (PKA) was obtained from Promega Corporation (Madison, Wisconsin). Adenosine 5’-triphosphate sodium salt (ATP) was obtained from Sigma Chemical

Cell 344

Company (St. Louis, Missouri). SPQ was obtained from Molecular Probes (Eugene, Oregon). [y”P]ATP was obtained from New England Nuclear (Boston, Massachusetts). Yeast nitrogen base without amino acids, Bacto-Peptone, and BactoAgar were obtained from Difco (Detroit, Michigan). Piaemid Constructions Plasmid RFG416 (a gift from Rick Gaber, Northwestern University) is a single copy CEN plasmid with the selectable marker UFtA3 and the pUC19 polylinker region. A 6.5 kb Sali-Sac1 fragment containing the STEG gene was subcloned from STE6-2~ (a gift from John McGrath, Massachusetts Institute of Technology) into the Sall and Sac1 sites of the vector RFG416 to produce plasmid JTSG. The yeast TRW gene (on a 0.65 kb Bglli-EcoRI DNA fragment) was inserted at nucleotide position 1356 of STEG NBDl, resulting in JTSGT. To construct the Hl STEG-CFTR hybrid plasmid, a 441 bp DNA fragment containing the CFTR NBDl region was synthesized using two STEG-CFTR oligonucleotide primers (primer 1, 5’~CCTTCGGAAGCAGTCCTGAAAGATAT-3’; primer 2, 5’-GATGAACAATATCTAGGTATCCAAAA-3’) and CFTR cDNA template DNA in a polymerase chain reaction (PCR) (Ho et al., 1969). PCR reactions were performed with a Temp-Tronic thermocycler (Barnsteadflhermolyne). Oligonucleotide primers 1 and 2 encoded fusion junctions of STEG L37.5 to CFTR K442 and CFTR L576 to STEG D537, respectively, found in the Hl STEG-CFTR hybrid gene. The CFTR portion of each primer is underlined. ST/X DNA flanking NBDl was added to each end of the 441 bp fragment by PCR, resulting in a 667 bp DNA fragment consisting of 419 bp of CFTR DNA (encoding CFTR amino acids K442L578) that is flanked at the 5’end by 168 bp of STEG DNA (encoding STEG amino acids K319-L375) and 280 bp of STEG DNA at the S’end (encoding STEG amino acids D537-G640). Plasmid construction of Hl was performed by cotransformation (ito et al., 1983) of yeast strain JPYPOl with the 867 bp DNA fragment and 3 ug of plasmid JTSGT and selection of transformants on SD-URA (yeast nitrogen base supplemented with all amino acidsexcept uracii). Homologous recombination between the STEG DNA sequences at each end of the 867 bp DNA fragment with the STEG gene on the plasmid results in the targeted integration(Orr-Weaveret al., 1981)oftheCFTRsequences intoNBD1 of STE6 on piasmid JTSGT and the consequent loss of the TRP 1 gene. Recombinants containing the desired STEG-CFTR hybrid gene were identified as rrp- auxoptrophs at a frequency of about 1% among the transformants. Plasmid DNA was prepared from trp- transformants, and the structure of the STEG-CFTR gene was confirmed by DNA sequencing analysis. STEG-CFTR hybrid genes H2-H6 were similarly constructed using the following oligonucleotide primers for constructing the appropriate STEG-CFTR junctions: STEG 0440/CFTR F494,5%ACCGTCGTAGAACAGTTTTCCTGGATTA-3’; CFTR G509/ STEG S457,5”CCGAATCTGTTGAACCAAAGATGATATTT-3’; CFTR G550ISTE6 G509,5”‘lTGTTGTTGCCCGCCACTCAGTGTGATTC-3’; CFTR R560/STE6 A519, S-ATCTCTGATGAATGCTCTTGCTAAAGAAAT-3’. Yeast Mating Assays The yeast strains JPYPOl (MATa, ste6kHIS3, ga12, ura3-52, /ys2-801, trp7, /eu2-3,112, his3 4200) and 22-2D (MATa, ura3-52, /eu2-3,112, trp7) were used for all mating experiments. JPY201 contains a STEG deletion (including NBDl and extending beyond the termination codon)and replacementwith theyeastH/S3gene(McGrathandVarshavsky, 1989). Quantitative mating assays were performed as in Trueheart et al. (1987). Transformants of JPYPOI containing each STEG-CFTR chimera were grown to log phase in 0.1% glucose SD-URA media. From each culture, 3 x lo6 cells were mixed with an equal number of 22-2D cells grown in YPD media and collected by filtration onto a Millipore filter, which was then placed upon a YPD plate for 4 hr at 30%. Cells were resuspended, sonicated briefly, and plated from serial dilutions onto SD+LEUTTRP (yeast nitrogen base supplemented with leucine and tryptophan). Diploid colonies were counted after 3 days at 30%. Control strains ST/Z6 wild-type and ste6A consisted of JPYPOl transformed with piasmids JTSG and JTSGT, respectively. For qualitative petri dish mating assays (Figure 2), JPYPOl transformants containing STEG-CFTR chimeras were grown as patches on SD-URA media and then replica printed to YPD media on a lawn of 22-20 cells,

Following incubation at 30°C for 8 hr, the plate containing cells was replica printed to SD+LEU/TRP and incubated 30°C to allow growth of diploid colonies.

the mating 3 days at

In Vitro Mutagenesis of H5-AF508 Plaemid To generate mutations at R553 of H5-AF508, complementary overlapping oligonucleotides were synthesized that contained random DNA sequence at the R553 codon. These oligonucleotides were used in the same PCR protocol as described above, using H5A plasmid DNA template, to generate a 867 bp DNA containing random mutations at the R553 locus. The PCR fragment DNA was cotransformed into yeast with pJTS6T DNA, and recombination of the fragment with plasmid gave rise to frp- transformants containing the H5-AF508 hybrid gene with a random mutation at R553. A total of seven rrp- recombinant transformants were isolated having a mutation at the R553 position that were then tested by the mating assay to identify those having a higher mating efficiency than H5-AF508 control. Two such colonies were identified (which contained the R553Q and R553M mutations), plasmid DNA was isolated from each, and the DNA sequence of the NED1 region was determined. The same protocol was used to generate random mutations at each of the positions 1556, S557, and L558 in the H5-AF508 plasmid. A total of 13 trp recombinant transformants were isolated from the mutagenesis of these three codons. None of the transformants had a higher mating efficiency than H5-AF508. No mutations were generated in codons A554 and R555 of H5-AF508. Mutations in CFTR expression plasmids were generated as previously described using oligonucleotide mutagenesis (Kunkel. 1985). Ceils and CFTR Expression Systems We used the vaccinia virus-T7 hybrid expression system developed by Moss and colleagues (Elroy-Stein et al., 1989). HeLa cells were maintained as previously described in Berger et al. (1991). To express CFTR in HeLa cells transiently, cells were infected with recombinant vaccinia virus (vTF7-3) to express the T7 bacteriophage RNA polymerase and then transfected with plasmid DNA containing either wild-type CFTR (pTM-CFTR-4) or CFTR mutants (pTMCFTRAF508, pTMCFTRAF508/R553Q, pTMCFTRAF508/R553M, pTMCFTR/R553Q, and pTMCFTWR553M) under the control of the T7 promoter, essentially as described in Rich et al. (1990). Plasmid-transfected HeLa cells were used for all experiments except for the data shown in Figure 48, in which HeLa cells were infected with two recombinant vaccinia viruses, one expressing either wild-type CFTR or the designated CFTR mutations and the other expressing T7 RNA polymerase (vTF7-3). Recombinant vaccinia virus was prepared as in Mackett et al. (1985). immunocytochemietry and Protein Analysis lmmunoprecipitations and antibody staining of CFTR in HeLa cells transfected with CFTR and CFTR mutants were performed as in Denning et al. (1992b). Pe&ch-Clamp Technique Patch-clamp recording are similar to those previously described (Hamill et al., 1981; Berger et al., 1993). The excised, inside-out configuration was used in all patch-clamp experiments. Cells and bath were maintained at 30°C-35% by a temperature-controlled microscope stage (Brook Industries, Lake Villa, Illinois). Pipette resistance was 2-6 MD and seal resistance was 2-25 GD. A List EPC-7 amplifier (Adams and List Associates, Limited, Westbury, New York) was used for current amplification and voltage clamping. A laboratory computer system (Indec Systems Incorporated, Sunnyvale, California) was used for data acquisition and analysis. Currents were filtered at 1 kHz and digitized at 2 kHz. Voltage was referenced to the external surface of the membrane patch: a depolarizing voltage is positive. The pipette (extracellular) solution contained 140 mM N-methyl-o-glucamine, 2 mM MgCI,, 5 mM CaCI,, 100 mM L-aspartic acid, and 10 mM HEPES (pH 7.3 with HCI) (Clf concentration, 50 mM). The bath (cytosolic) solution contained 140 mM N-methyl-o-glucamine, 3 mM MgCb, 1 mM cesium ethylene glycol-bis(j3-aminoethyl ether)n,n,n’,n’-tetraacetic acid (CsEGTA), and 10 mM HEPES (pH 7.3 with HCI) (Cl- concentration, 140 mM). The estimated free Cal+ concentration in the internal solution was
Revertants 345

of CFTRAF508

across the membrane patch, we were able to check frequently the current reversed as expected for a Cl--selective channel.

that

Statistical Analysis The P. and single-channel conductance were determined from amplitude histograms. The P. was measured in patches containing <5channels. The number of channels was determined from the maximum number of channels open simultaneously. Values are presented as the mean + SEM. The unpaired Student’s t test was used when comparing values. Acknowledgments We thank J. Riordan for providing laboratory space for J. L. T. at the Hospital for Sick Children; J. McGrath. R. Gaber, and J. Rommens for providing plasmids and strains; and M. Keene, A. Puga, D. Ries, L. deBerg, and D. Petersen for technical assistance. This research was supported by a grant from the Cystic Fibrosis Foundation (CFF) and by the Howard Hughes Medical Institute (HHMI). J. L. T. is a recipient of a CFF postdoctoral fellowship. H. A. B. was supported by a Parker B. Francis Fellowship Award. L.-C. T. is Seller Chair in Cystic Fibrosis Research, a Scientist of the Medical Research Council of Canada, andan InternationalScholarof HHMI. M. J. W. isan Investigator of the HHMI. Received

January

20, 1993; revised

February

9, 1993.

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