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Originally published In Press as doi:10.1074/jbc.C600209200 on August 22, 2006

J. Biol. Chem., Vol. 281, Issue 40, 29436-29440, October 6, 2006
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Insertion of an Arginine Residue into the Transmembrane Segments Corrects Protein Misfolding*Formula

Tip W. Loo, M. Claire Bartlett, and David M. Clarke1

From the Department of Medicine and Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Received for publication, August 4, 2006 , and in revised form, August 16, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Deletion of Phe-508 ({Delta}F508) in cystic fibrosis transmembrane conductance regulator causes cystic fibrosis because of misfolding of the protein. P-glycoprotein (P-gp) containing the equivalent mutation ({Delta}Y490) is also misfolded but can be rescued with drug substrates. Whether rescue is due to direct binding of drug substrate to the transmembrane (TM) segments or to indirect effects on cellular protein folding pathways is still controversial. P-gp-drug substrate interactions likely involve hydrogen bonds. If the mechanism of drug rescue involves changes to TM packing then we should be able to identify suppressor mutations in the TM segments that can mimic the drug rescue effects. We predicted that an arginine residue in the TM segments predicted to line the drug-binding pocket of P-gp (I306(TM5) or F343(TM6)) might suppress {Delta}Y490 P-gp protein misfolding because it has the highest propensity to form hydrogen bonds. We show that R306(TM5) or R343(TM6) increased the relative amount of mature {Delta}Y490 P-gp by 6-fold. Most other changes to Ile-306 or Phe-343 did not enhance maturation of {Delta}Y490 P-gp. The I306R mutant also promoted maturation of misprocessed mutants that had mutations in the second nucleotide-binding domain (L1260A), the cytoplasmic loops (G251V, F804A), the linker region (P709A), or in TM segments (G300V, G722A). These results show that arginine residues in the TM domains can mimic the drug rescue effects and are effective suppressor mutations for processing mutations located throughout the molecule.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cystic Fibrosis (CF)2 is a lethal inherited disorder caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein (1). The most common CF-associated mutation is deletion of Phe508 ({Delta}F508) in the first nucleotide-binding domain (NBD1) (2). The mutation causes protein misfolding. The misfolded protein is retained in the endoplasmic reticulum and is rapidly degraded (3, 4). The equivalent deletion in human P-glycoprotein ({Delta}Y490) also leads to defective folding and rapid degradation (5).

The presence of the {Delta}F508 mutation in CFTR has little effect on the conformation of NBD1, its in vitro stability, or folding kinetics (6). The primary effect of {Delta}F508 is disruption of NBD1-TMD1 interactions. In P-gp, the {Delta}Y490 mutation also disrupts NBD1-TMD1 interactions (7). Disruption of NBD1-TMD1 interactions then appears to disrupt subsequent interactions between TMD1 and TMD2 (8, 9).

An important clinical goal in CF is to specifically correct the folding defect in {Delta}F508-CFTR as the mature protein still has cAMP-activated chloride channel activity (10). Studies on the {Delta}Y490 P-gp model system suggest that specific drug interactions with the TM segments can promote maturation of the mutant protein. Therefore, the results imply that the TM segments of {Delta}F508-CFTR would be a good target for the development of pharmacological chaperones. The mechanism of how pharmacological chaperones rescue folding defects in P-gp (11) is controversial because it is difficult to distinguish whether increased maturation was due to direct effects on TM packing (12) or due to indirect effects on cellular folding pathways. For example, stress-induced alterations in molecular chaperone levels (13–15) or endoplasmic reticulum-associated degradation (16) could potentially promote maturation of {Delta}F508 CFTR or {Delta}Y490 P-gp. Therefore, it is important to demonstrate that manipulation of the TM segments can promote folding of mutant proteins in the absence of potential stress-induced cell responses.

In this study, we tested whether mutations introduced in the TM segments forming part of the drug-binding pocket could promote maturation of {Delta}Y490 Pgp.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of Mutants—Two tagged versions of human P-gp were used in this study. A P-gp cDNA contained the epitope for monoclonal antibody A52 at the COOH-terminal end was used to distinguish the expressed protein from any endogenous P-gp (17). The second P-gp cDNA was modified to contain a 10-histidine tag at the COOH-terminal end to facilitate purification of the expressed protein by nickel-chelate chromatography (18). Mutations were introduced into wild-type P-gp cDNAs and {Delta}Y490 P-gp cDNAs as described previously (7, 19). The integrity of all the mutant cDNAs was confirmed by sequencing the entire cDNA (20).

Expression and Detection of Mutants—The mutant P-gps were transiently expressed in human embryonic kidney (HEK) 293 cells in plain media as described previously (18). Whole cell SDS extracts were subjected to immunoblot analysis with monoclonal antibody A52 (21). Digestion with endoglycosidase H or peptide:N-glycosidase F (PNGase F) (New England Biolabs, Mississauga, Ontario, Canada) and cell surface labeling of P-gp with biotin-LC-hydrazide (Invitrogen, Burlington, Ontario, Canada) were performed as described previously (21, 22).

Purification of P-gp and Measurement of ATPase Activity—Histidine-tagged P-gp was expressed in HEK 293 cells and then isolated by nickel-chelate chromatography as described previously (18). A sample of the isolated histidine-tagged P-gp was mixed with an equal volume of 10 mg/ml sheep brain phosphatidylethanolamine (Type II-S, Sigma) that had been washed and suspended in Tris-buffered saline. The sample was sonicated and ATPase activity measured in the presence of various concentrations of verapamil or colchicine.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Mutations in the TM segments, cytoplasmic loops connecting the TM segments, the two NBDs, and within the linker region connecting the two halves of P-gp inhibit maturation of the protein (processing mutations) (Fig. 1A). Residue Tyr-490 in P-gp is equivalent to Phe-508 in CFTR (5). Deletion of Tyr-490 in P-gp also inhibits maturation of the protein (7). These mutants can be rescued, however, by carrying out expression in the presence of a P-gp drug substrate such as cyclosporin A, vinblastine, or verapamil (11). We had proposed that drug substrates rescue misfolded P-gp mutants through interactions with the TM segments (12). It is possible, however, that drug substrates have indirectly altered the general cellular protein pathways since the drug substrates can have pleiotropic effects on cells.

If drug substrates can promote maturation of P-gp mutants (drug rescue) by occupation of the drug-binding site (where it acts as a scaffold during biosynthesis of the protein), then it should be possible to introduce suppressor mutations in the drug-binding pocket of P-gp that would mimic the drug rescue effects. Hydrophobic residues or residues that can form hydrogen bonds would be predicted to promote protein maturation since it has been postulated that drug substrate/P-gp interactions appear to be mediated by hydrophobic interactions and hydrogen bonding (23). The number and strength of hydrogen bonds are important determinants of P-gp-drug substrate interactions (24, 25).

Residue I306(TM5) (Fig. 1A) was initially selected for mutational analysis because cysteine mutagenesis and thiol-labeling studies indicated that it lies within the drug-binding pocket of P-gp. It was found that modification of Cys-306 with a thiol-reactive analog of verapamil permanently activated the ATPase activity (26). In addition, mutations to Ile-306 can decrease the affinity of P-gp for some substrates by up to 50-fold (27). Accordingly, mutations were made to Ile-306 in the {Delta}Y490 P-gp. Residue Ile-306 was changed to a positively charged (Arg), a negatively charged (Glu), polar (Gln, Ser), a large non-polar residue (Trp), or to relatively small (Ala, Gly) residues. The mutants were expressed in HEK 293 cells and whole cell SDS extracts subjected to immunoblot analysis. Fig. 1B shows that very little (<10%) of the mature product (170 kDa) was present in cells transfected with mutant I306/{Delta}Y490 P-gp cDNA. By contrast, the I306R mutation caused about a 6-fold increase in mature (170 kDa) protein. Smaller increases in the mature protein were observed with the I306Q (about 3-fold) and the I306W (about 2-fold) mutants. No increase in the amount of mature protein (170 kDa) was detected for mutants I306S, I306A, I306E, or I306G. These results show that introduction of an arginine at position I306 was the most efficient change that promoted maturation of mutant {Delta}Y490 P-gp.


Figure 1
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  		FIGURE 1.
Schematic model of P-gp. A, the 12 TMs of P-gp are shown as numbered cylinders. The locations of the processing mutations (gray circles) and residues Ile-306 and Phe-343 (black circles) are shown. The branched and zigzag lines represent glycosylation sites and the linker region, respectively. TMD and NBD represent the transmembrane and nucleotide-binding domains, respectively. B, immunoblot analysis of A52-tagged wild-type (WT) or {Delta}Y490 Pgp mutants with the indicated changes to residue I306(TM5). C, the amount of mature P-gp relative to total (mature 170 kDa plus immature 150-kDa protein) was quantitated by scanning the gel lanes followed by analysis with the NIH Image program (available at rsb.info.nih.gov.nih-image/) and an Apple Macintosh computer. The experiments were done in quadruplicate. D, immunoblot analysis of whole cell SDS extracts of HEK 293 cells transfected with A52-tagged {Delta}Y490 Pgp cDNAs containing an arginine at various positions in TM5 (positions 304–308). The positions of the mature (170 kDa) and immature (150 kDa) P-gps are indicated.

 
We then tested whether the presence of an arginine at other positions in TM5 would promote maturation of mutant {Delta}Y490 P-gp. An arginine residue was introduced at positions 304, 305, 307, or 308 in TM5 in the {Delta}Y490 P-gp mutant. The mutants were expressed in HEK 293 cells and whole cell extracts subjected to immunoblot analysis. Fig. 1D shows that only the I306R mutation promoted maturation of the {Delta}Y490 P-gp mutant.


Figure 2
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  		FIGURE 2.
Effect of I306R on maturation of processing mutants. A, immunoblot analysis of whole cell extracts of HEK 293 cells expressing the A52-tagged P-gp processing mutants G251V, G300V, P709A, G722A, F804A, and L1260A with or without the I306R mutation. B, HEK 293 cells expressing A52-tagged mutants G251V or G251V/I306R were treated with endoglycosidase Hf (H), PNGase F (F), or no endoglycosidase (–). Equivalent amounts of samples were subjected to SDS-PAGE on 6.5% gels and immunoblot analysis. The positions of the mature (170 kDa), immature (150 kDa), and unglycosylated (140 kDa) P-gps are indicated. C, HEK 293 cells mock-transfected (Control) or transfected with A52-tagged mutant G251V or mutant G251V/I306R were subjected to cell surface labeling with biotin-LC-hydrazide. Biotinylated P-gp was immunoprecipitated with monoclonal antibody A52 and subjected to analysis with streptavidin conjugated to horseradish peroxidase. The positions of the mature (170 kDa) and immature (150 kDa) P-gps are indicated.

 
The next step was to test whether the most effective suppressor mutation (I306R) of {Delta}Y490 P-gp could promote folding of other P-gp processing mutations. The I306R mutation was introduced into mutants containing a processing mutation in the TM segments (G300V in TM5, G722A in TM7), the loops connecting the TM segments (G251V in intracellular loop 2, F804A in intracellular loop 3), the linker region (P709A), and NBD2 (L1260A) (Fig. 1A). The mutants were expressed in HEK 293 cells and whole cell extracts were subjected to immunoblot analysis. Fig. 2A shows that I306R promoted maturation of all misprocessed mutants. The I306R mutation was more efficient, however, in promoting maturation of mutants G251V, G300V, P709A, and G722A than mutants F804A and L1260A.

Mutants G251V and G251V/I306R were then subjected to endoglycosidase digestion to see if the increased amount of 170-kDa protein was mature P-gp. Fig. 2B shows that the immature 150-kDa protein of mutant G251V was sensitive to both endoglycosidase H and PNGase F. Similarly, the 150-kDa G251V/I306R mutant protein was also sensitive to digestion by both endoglycosidases. The 170-kDa mature protein of mutant G251V/I306R, however, was only sensitive to PNGase F. These results indicate that the 170-kDa protein must have exited the endoplasmic reticulum and traversed the Golgi apparatus.

To determine whether the mature 170-kDa protein was at the cell surface, cells expressing A52-tagged mutant G251V or G251V/I306R were subjected to cell surface biotinylation with biotin-LC-hydrazide. The biotinylated P-gps were immunoprecipitated with monoclonal antibody A52. The immunoprecipitated P-gps were subjected to SDS-PAGE, transferred onto a sheet of nitrocellulose, and detected with streptavidin conjugated to horseradish peroxidase. Fig. 2C shows that the mature 170-kDa protein was detected at the cell surface only in mutant G251V/I306R. Introduction of I306R into other processing mutants such as {Delta}Y490 P-gp also increased cell surface expression of P-gp (data not shown). These results indicate that introduction of the I306R mutation results in increased expression of mature P-gp at the cell surface.


Figure 3
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  		FIGURE 3.
Effect of changes to residue Phe-343 on maturation of {Delta}Y490 P-gp. A, immunoblot analysis of whole cell SDS extracts of HEK 293 cells expressing A52-tagged {Delta}Y490 P-gp with the indicated mutation at position 343. B, the relative amount of mature P-gp to total P-gp (mature plus immature) in quadruplicate experiments were quantitated as described in the legend to Fig. 1B. C, immunoblot analysis of whole cell SDS extracts of HEK 293 cells expressing A52-tagged {Delta}Y490 P-gp containing I306, I306R, F343R, both I306R/F343R. The positions of the mature (170 kDa) and immature (150 kDa) P-gps are indicated.

 
The next step was to test whether introduction of an arginine mutation at another site in the drug-binding pocket would promote maturation of the {Delta}Y490 P-gp mutant. A potential site would be Phe-343 in TM6. Residue Phe-343 also lines the drug-binding pocket because mutant F343C could be labeled with the thiol-reactive drug substrate methanethiosulfonate-rhodamine to activate P-gp ATPase activity (28). Accordingly, {Delta}Y490-P-gp mutants were constructed in which Phe-343 was replaced with Trp, Arg, Ala, Ser, Tyr, Gly, Asn, Ile, or Asp. The mutants were expressed in HEK 293 cells and whole cell extracts were subjected to immunoblot analysis, Fig. 3A shows that introduction of an Arg mutation at position 343 significantly increased (about 6-fold) the amount of mature P-gp compared with mutant F343/{Delta}Y490 P-gp. Increased maturation efficiency was also observed in mutant F343N (about 3-fold), while smaller increases (1.5–2-fold) were observed with Trp or Tyr. Although mutant F343Y showed a small increase in the relative amount of mature protein, it also promoted degradation of the protein (seen as a product of about 130 kDa). The 130-kDa protein appears to be a degradation product that is found with P-gp mutants with mutations in TM6 (29).

To further test whether Phe-343 does reside within the drug-binding pocket of P-gp, we determined whether the F343R mutation altered the affinity of wild-type P-gp for substrates. Interaction of purified P-gp with drug substrates such as verapamil and colchicine can be monitored by stimulation of ATPase activity. Wild-type P-gp showed about a 21-fold maximal activation with verapamil with half-maximal activation (A50) at 46 µM verapamil (supplemental Fig. 1). The mutant F343R showed a marked reduction (69%) in maximal activity and a 21-fold decrease in apparent affinity for verapamil (A50 of 960 µM) (data not shown). The mutant also showed about a 70% reduction in maximal colchicine-stimulated ATPase activity compared with wild-type P-gp but no significant change in apparent affinity (A50 of about 1 mM for wild-type and mutant F343R). Therefore, the F343R mutation appears to reduce P-gp/verapamil interactions as well as coupling of colchicine or verapamil binding to stimulation of ATPase activity. Previous studies on mutant F343C by disulfide cross-linking analysis (30) or fluorescence labeling (31) have shown that Phe-343 is involved in conformational changes associated with coupling of drug binding to ATP hydrolysis.

Since the I306R and F343R mutations were the most efficient changes that promoted maturation of the {Delta}Y490 P-gp, we then tested whether the presence of both mutations would have an additive effect. Arginine residues were introduced at positions 306 and 343 in the {Delta}Y490 P-gp mutant and the I306R/F343R {Delta}Y490 P-gp mutant was expressed in HEK 293 cells. Whole cell SDS extracts were subjected to immunoblot analysis. Fig. 3C shows introduction of two arginine residues in different TMs of {Delta}Y490 P-gp increased the yield of mature enzyme (8.8-fold in mutant I306R/F343R {Delta}Y490 P-gp versus 6–6.3-fold in mutants I306R/{Delta}Y490 P-gp or F343R/{Delta}Y490 P-gp).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The results show that introduction of arginine mutations into the TM segments can suppress folding defects caused by processing mutations located throughout P-gp. An implication of the results is that the TM segments are ideal targets for pharmacological chaperones to promote folding of misfolded membrane proteins such as P-gp and CFTR. The TM segments may be particularly sensitive to the effects of processing mutations because packing of the TM segments may be the last step in the folding of ABC transporters (9). In vitro translation studies on CFTR with semipermeabilized cells in combination with limited proteolysis revealed that the cytosolic domains acquired their structures cotranslationally whereas post-translational folding only involved the TM domains (9). Mutations such as {Delta}Y490 in P-gp and {Delta}F508 in CFTR appear to interfere with post-translational folding of the proteins by disrupting NBD1-TMD1 interactions that are required for subsequent TMD1 and TMD2 interactions (6–8).


Figure 4
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  		FIGURE 4.
Models of the effect of drug substrate or the I306R mutation on maturation of {Delta}Y490 P-gp. A, deletion of Tyr-490 in NBD1 (X) inhibits protein folding by reducing the efficiency of NBD1-TMD1 interactions that then disrupts subsequent interactions between TMD1-TMD2. The protein accumulates in the cell in a partially folded conformation. B, drug substrate promotes maturation through many H-bond (dotted lines) interactions with the TM segments and acts as a scaffold during folding. C, the presence of Arg at position 306 mimics the effects of drug substrate by promoting packing of the TM segments by H-bond formation (dotted lines) with residues in TMD2 resulting in maturation of {Delta}Y490 P-gp.

 
Maturation of the {Delta}Y490 P-gp mutant was promoted by introducing mutations into the TM segments forming the drug-binding pocket. An arginine at positions 306(TM5) or 343(TM6) acted as the most effective suppressor mutation. The ability of an arginine residue in a TM segment to promote protein folding may be unexpected because it has been reported that the most common disease-causing mutation in membrane proteins is due to the presence of arginine in the TM segments (32). Arginine in the TM segments may promote maturation of P-gp processing mutants because its side chain contains three polar atoms and therefore has the potential to form three H-bonds with other TM segments. It is unlikely that the introduced arginine residues form ionic interactions with acidic groups because there are no Glu or Asp residues in the TMs of P-gp. The ability of arginine in the TM segments to form a H-bonded network between the TM segments has been reported for bovine rhodopsin (32, 33).

Since there is evidence that drug substrates promote interactions between the two TMDs of P-gp (12), we would predict that the I306R or F343R mutations also promote interactions between the TMDs through H-bond formation. Fig. 4 shows a model of {Delta}Y490 P-gp folding. The NBDs would fold cotranslationally but the presence of the {Delta}Y490 mutation interferes with formation of the NBD1-TMD1 contacts. This in turn would disrupt proper packing of the TM segments (Fig. 4A) resulting in a protein that is trapped in a prefolded and inactive conformation (34). The presence of drug substrate (Fig. 4B) or I306R or F343R mutations (Fig. 4C) would promote post-translational packing of the TM segments. A reason that the drug substrates are better than arginine at promoting maturation of {Delta}Y490 P-gp (>10-fold increase in mature protein with cyclosporin A (11)) could be due to the larger number of contacts (or H-bonds) that drug substrates could make with the TM segments (25) compared with an arginine side chain. Therefore, drug substrates would be better as scaffolds for stabilizing the mutant intermediate during biosynthesis than arginine.

Another possible explanation for the observed results is that the introduced arginines mimic low temperature rescue by simply slowing protein folding to allow for the system to sample different folding pathways. It appears, however, that different mechanisms must operate during rescue by the introduced arginines compared with rescue by low temperature incubation. This is supported by the observation that maturation of mutant G251V is temperature-insensitive (very little mature P-gp is detected after expression for 48 h at 26 °C) (17), but introduction of the I306R mutation caused about an 8-fold increase in mature P-gp (Fig. 3). In addition, the I306R mutation did not slow folding of wild-type P-gp (27). Similar results were observed with mutant F343R in the wild-type background (data not shown).

In summary, the results show that arginines in the TM segments are effective suppressor mutations that mimic the drug rescue effects and can promote maturation of a mutant ABC transporter that has a processing mutation in any domain of the molecule.


   FOOTNOTES
 
* This work was supported by grants from the National Cancer Institute of Canada through the Canadian Cancer Society and from the Canadian Institutes of Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1. Back

1 Recipient of the Canada Research Chair in Membrane Biology. To whom correspondence should be addressed: Dept. of Medicine, University of Toronto, 1 King's College Circle, Rm. 7342, Medical Sciences Bldg., Toronto, Ontario M5S 1A8, Canada. Tel.:/Fax: 416-978-1105; E-mail: david.clarke{at}utoronto.ca.

2 The abbreviations used are: CF, cystic fibrosis; ABC, ATP-binding cassette; CFTR, cystic fibrosis transmembrane conductance regulator; P-gp, P-glycoprotein; NBD, nucleotide-binding domain; HEK, human embryonic kidney; TM, transmembrane; TMD, transmembrane domain; PNGase F, peptide:N-glycosidase F. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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