RESULTS

Mutation of ATP6V1B1

Genomic DNA sequence analysis in a large, previously phenotypically characterized Mexican family with severe distal renal tubular acidosis and deafness¹⁰ revealed a novel frameshift mutation in exon 14 of the ATP6V1B1 gene resulting from a GT duplication at position 1401 (c.1401_ 1402dupGT). Homozygosity of this mutation segregates completely with classical distal renal tubular acidosis in the kindred. In contrast to all the reported ATP6V1B1 mutations that are located in the first two-thirds of the B1 subunit, the novel mutation results in a truncation near the C-terminus and insertion of 19 amino acids (p.Phe468fsX487) (Figure 1).

Figure 1.

A novel ATP6V1B1 frameshift mutation. Wild-type and mutant (c.1401_ 1402dupGT) nucleotide sequences of exon 14 are shown in the upper panel. Alignment of C-terminal amino-acid sequences of human wild-type (WT) and mutant (p.Phe468fsX487; M) B1, human B2, and S. cerevisiae Vma2p subunits. The frameshift-induced amino-acid change is shown in red, and the B1 subunit C-terminal PDZ-binding motif (DTAL) is shown in blue (lower panel).

Full figure and legend (65K)

Identification of V₁ domain assembly disruption as the molecular mechanism for the novel disease-causing mutation

As the V-ATPase undergoes complex assembly and processing,^{2, 11} we examined V₁ domain assembly as a potential molecular basis of dysfunction. We expressed epitope-tagged human wild-type and disease-associated p.Phe468fsX487 (mutant) B1 subunits in distal tubule-like Madin–Darby canine kidney (MDCK) cells. Wild-type but not mutant B1 subunits, co-immunoprecipitated with the endogenous V₁ domain E subunit in MDCK cells, demonstrating assembly of the wild-type but not the mutant B1 subunit into the V₁ domain (Figure 2a). Identical results were obtained when the constructs were expressed in a different host cell line (human embryonic kidney cell line (HEK293)) or when the epitope-tag was relocated (N-terminal FLAG versus C-terminal anti-hemagglutinin (HA)) (Figure 2a), so the results are independent of host or tag locale. With the exception of a class I postsynaptic density 95/discs large/zonula occludens-1 (PDZ)-binding motif^{14, 15} at the C-terminus of the B1 subunit, little is known about the function of the last 30 amino acids of mammalian V-ATPase B subunits.^{16, 17} Two studies employing cysteine cross-linking of Saccharomyces cerevisiae V-ATPase V₁ domain subunits have identified N- and C-terminal residues (Glu 106/Asp 199 and Glu 494/Thr 501, respectively) to be important for the interaction with the E subunit.¹⁸ These residues are predicted to reside on the outer surface of V-ATPase B subunit (Figure 3), near both the top and the bottom of the V₁ domain.¹⁸ Glu 494 and Thr 501 of S. cerevisiae V-ATPase B subunit, however, are in areas that lack homology to mammalian V-ATPase B subunits (Figure 1). As the mutation abolishes the PDZ-binding motif in the B1 subunit, we tested if deletion of this region could account for the defective V-ATPase assembly, possibly via disruption of PDZ interaction. As shown in Figure 2a, disruption of the PDZ-binding motif (DTAL) by truncation of three amino acids (-TAL) completely eliminated interaction of the B1 subunit with the PDZ protein NHERF-1, but the ability to integrate into a V₁ domain remains completely intact. Thus, truncation of the PDZ domain is insufficient to disrupt V₁ assembly.

Figure 2.

Detailed analysis of the p.Phe468fsX487 mutation. (a) Co-immunoprecipitation in MDCK cells transiently co-expressing C-terminal HA-tagged NHERF-1 and N-terminal FLAG-tagged B1 subunit constructs (WT, wild-type, M, mutant, -TAL, 3 amino-acid truncation, left panel). Immunoprecipitation (IP) was performed with an -FLAG antibody; immunoblotting (IB) with antibodies is shown. -E is against the native E subunit.¹² In the right panel, HEK293 cells were transfected with N-terminal FLAG- or C-terminal HA-tagged B1 subunit. Immunoprecipitation was performed with either -FLAG or -HA antibodies and immunoblotting with depicted antibodies. (b) HEK293 cells and MDCK cells were transiently transfected with wild-type (WT), mutant (M), and a common polymorphic variant p.Glu161Lys (E161K) of B1. Cells were broken with Polytron in the total absence of detergent and the supernatants were retrieved after 50 000 g centrifugation and immunoblotted with the indicated antisera. (c) MDCK cells transiently expressing wild-type (WT) or mutant (M) N-terminal FLAG-tagged B1 subunits after 8 h of cycloheximide chase (40 g ml^{- 1}). Cells were labeled with a primary -FLAG antibody and a secondary antibody conjugated to Texas Red (red) and counter-stained with DAPI (blue). (d) S. cerevisiae Vma2p-null strain, expressing C-terminal HA-tagged constructs in a p426TEF expression vector,¹³ grown on pH-7.5 YEPD plates for 5 days. A total of 5 10⁴ cells were plated with serial 10-fold dilutions (left to right). The right panel shows the relative levels of expression of the constructs by immunoblot detected with an -HA antibody.

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Figure 3.

Structural model of human V-ATPase B1 subunit. The model was constructed with the Swiss-PdB viewer using the X-ray crystal structure of bovine F-ATPase subunit as template and coordinates obtained from the Protein Data Bank (accession number 1bmfE).¹⁹ The structure is oriented such that the region farthest from the membrane is at the top, the external surface of the V₁ domain is shown on the right, whereas the region of the B1 subunit oriented toward the interior of the V₁ domain is shown in the left. Point mutations used in this study are shown in red and C-terminus truncations are shown in black. C- and N-terminal amino-acid residues are missing because of a lack of homology to the bovine F-ATPase subunit.

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The disruption of V₁ assembly was further demonstrated by analysis of a nondetergent supernatant of transiently transfected cells, so there is no possibility of release of V₁ or any of its subunits from lipid bilayer (Figure 2b). This supernatant represents V₁ subunits in the cytosol before their insertion into lipid membrane. In both HEK293 and MDCK cells, the mutant B1 subunit in the cytosol (preinsertion into membrane) failed to co-precipitate with subunits E, D, or A. Premembrane insertion V₁ assembly was intact in wild-type and a previously described polymorphic variant of B1 (p.Glu161Lys) (Figure 2b).

We next localized wild-type or mutant B1 subunits by immunocytochemistry. MDCK cells overexpressing wild-type or mutant B1 subunits were treated with cycloheximide (40 g ml^{- 1}) for 8 h to chase expressed tagged proteins to their final location (Figure 2b). Under laser confocal microscopy, wild-type B1 subunit demonstrated a characteristic intracellular 'vesicular-appearing' pattern, suggesting incorporation into the V-ATPase into intracellular membranous compartments, compatible with our biochemical findings. The identity of these compartments are not known at present. In contrast, the mutant B1 subunit showed a diffuse cytoplasmic and interestingly nuclear staining.

Functional assessment of the novel B1 subunit mutation in yeast

Because of the omnipresence of V-ATPase B subunits in mammalian cells, we deployed a S. cerevisiae strain devoid of the endogenous yeast B subunit (Vma2p) for definitive functional assessment of wild-type and mutant human B1 subunits without contamination from native B subunits. The Vma2p-null strain grows at acidic pH but fails to grow at neutral or alkaline pH. Growth at neutral or alkaline pH can be rescued by expression of Vma2p.^{16, 17, 20} Figure 2c shows transformed yeast in a dilution series on a pH-7.5 yeast extract peptone dextrose (YEPD) agar plate after growth for 4 days at 25°C.²¹ Complementation of the deleted endogenous yeast Vma2p subunit (growth at pH 7.5) was observed in yeast-expressing wild-type but not mutant human B1 subunits. Identical results were obtained for untagged expression constructs and chimeric constructs where the C-terminus of the yeast Vma2p subunit was replaced with a human wild-type or mutant B1 subunit C-terminus (data not shown). In addition, no dominant-negative effect was observed when wild-type and mutant human B1 subunits were co-expressed (data not shown), which is consistent with the lack of an obvious clinical phenotype in the heterozygous individuals.

Growth at pH 5.5 was the same for all constructs tested and no toxic effects of individual constructs were observed (data not shown). Growth of transformed yeast-expressing wild-type human B1 subunit requires functional assembly of V₀ and V₁ domains. As V₁ domain assembly is a stepwise process,¹¹ biochemical evidence of B1 subunit interactions with other V₁ domain subunits does not guarantee full V₁ domain assembly. The yeast data, however, demonstrate that the human wild-type B1 subunit constructs assemble into complete and functional V₁ domains.

The B1 subunit C-terminus is critical for interaction with the E subunit

To further delineate the importance of the B1 subunit C-terminus in V-ATPase assembly, we generated serial C-terminus truncations (Figure 3) and analyzed both V₁ domain assembly in HEK293 cells and functional complementation in yeast. At equal expression levels in HEK293 cells (Figure 4a, upper panel), truncations of the B1 subunit 510 (-TAL), 501, and 490 still allow V₁ domain assembly (Figure 4a, lower panel). Whereas the interaction with the endogenous E subunit is weak in truncation 490 compared with wild type, the 501 truncation seems to enhance the interaction. When expressed in MDCK cells, wild-type B1 displayed a vesicular pattern and p.Phe468fsX487 mutant shows the abnormality as described earlier (Figure 4b). Although 510, 501, and to a certain extent 490 still exhibit wild-type morphology, further truncations led to a complete loss of the pattern seen in wild-type B1.

Figure 4.

Analysis of C-terminus truncations of the human V-ATPase B1 subunit. (a) HEK293 cells expressing N-terminal FLAG-tagged B1 subunit truncations (upper panel). A total of 40 g of lysate protein were loaded and immunoblotting (IB) was performed using an -FLAG antibody. The lower panel shows co-immunoprecipitation of endogenous E subunit after immunoprecipitation of B1 subunit truncations with an -FLAG antibody in HEK293 cells. (b) MDCK cells were transiently transfected with N-terminal FLAG-tagged wild-type (WT), mutant (M), and various C-terminal truncations of B1. After 8 h of cycloheximide chase (40 g ml^{- 1}), cells were labeled with a primary -FLAG antibody and a secondary antibody conjugated to Texas Red (red) and counter-stained with DAPI (blue). (c) S. cerevisiae Vma2p-null strain expressing C-terminal HA-tagged B1 subunit truncation constructs. A total of 5 10⁴ cells were plated with serial 10-fold dilutions (top to bottom) on a pH-7.5 YEPD plate, and growth was assessed after 5 days (lower panel). The upper panel shows expression of the truncation constructs in yeast by immunoblot detected with an -HA antibody. Equal amounts of yeast lysates from 1 o.d. unit were loaded.

Full figure and legend (158K)

When tested for functional complementation in the Vma2p-null strain, 510 and 501 support growth at alkaline pH comparable with the wild-type B1 subunit, whereas growth of 490 is considerably weaker (Figure 4c, lower panel), a finding that is in concordance with the weaker V₁ domain assembly in HEK293 cells. Expression levels of truncated B1 subunits in yeast were equal for all constructs (Figure 4c, upper panel). Thus, truncations beyond amino acid 490 of the B1 subunit disrupt V₁ domain assembly and V-ATPase function. Figure 1 shows the high level of conservation of amino acids preceding truncation 490. These results demonstrate the importance of the B1 subunit C-terminus in V-ATPase assembly. Structural homology modeling with the X-ray crystal structure of bovine F-ATPase subunit predicts an -helix between amino-acid residues 490 and 505 of the B1 subunit (Figure 3). Our results support a pivotal role of this very C-terminal -helix in the association between E and B1 subunit in the V₁ domain. This hypothesis is supported by the fact that not the length per se but the specific amino-acid sequence of the C-terminus is important for interaction with the E subunit, as a C-terminal HA-tagged mutant B1 subunit with a total of 495 amino acids also fails to assemble (Figure 1).

Molecular mechanism for disease-causing B1 subunit mutations

Having identified lack of V₁ domain assembly and consecutive loss of function as a mechanism of a human V-ATPase-associated disease, the question arises if assembly failure is a common underlying mechanism of ATP6V1B1 mutations. We therefore analyzed all described ATP6V1B1 missense mutations: c.232G>A (p.Gly78Arg); c.242T>C (p.Leu81Pro); c.368G>T (p.Gly123Val); c.370C>T (p.Arg124Trp); c.469C>T (p.Arg157Cys); c.521T>G (p.Met174Arg); c.823A>C (p.Thr275Pro); c.947G>A (p.Gly316Glu); c.1037C>G (p.Pro346Arg) and c.1090G>A (p.Gly364Ser) and two frequent SNPs c.89C>T (p.Thr30Ile) and c.481G>A (p.Glu161Lys) that have previously been described to be in Hardy–Weinberg equilibrium in control populations and believed to have no functional impact on pump function.^{7, 8, 22} Figure 3 shows the location of the mutations and SNPs in the putative B1 subunit structure. There are additionally reported nonsense and frameshift mutations leading to early stop codons in the transcript, likely to result in severely truncated B1 subunits and were thus not included in our analysis. As shown in the lower panel of Figure 5a, all missense base changes fail to interact with the E subunit except p.Thr30Ile, p.Glu161Lys, and p.Gly316Glu, which all retain efficient V₁ domain assembly. Mutant and wild-type B1 subunit constructs were expressed at equal levels in HEK293 cells (Figure 5b).

Figure 5.

Analysis of ATP6V1B1 missense mutations. (a) HEK293 cells expressing N-terminal FLAG-tagged B1 subunit point mutations (upper panel). A total of 40 g of lysate protein were loaded and immunoblotting (IB) was performed using an -FLAG antibody. The lower panel shows co-immunoprecipitation of endogenous E subunit after immunoprecipitation of B1 subunit truncations with an -FLAG antibody in HEK239 cells. (b) S. cerevisiae Vma2p-null strain expressing C-terminal HA-tagged B1 subunit point mutation constructs. A total of 5 10⁴ cells were plated with serial 10-fold dilutions (top to bottom) on a pH-7.5 YEPD plate and growth was assessed after 5 days (lower panel). The upper panel shows expression of the point mutation constructs in yeast by immunoblot detected with an -HA antibody. Equal amounts of yeast lysates from 1 o.d. unit were loaded.

Full figure and legend (86K)

All reported missense mutations except one (p.Thr30Ile) fail to support growth of transformed yeast at alkaline pH, further validating our functional assay in yeast. The point mutant p.Thr30Ile was similar to wild-type B1 subunit, suggesting that this is a fully functional polymorphic allele. In contrast, p.Glu161Lys exhibits poor growth at alkaline pH, suggesting that this is not a silent SNP but possibly a dysfunctional missense mutation. In contrast to Thr 30, Glu 161 is conserved in animals, fungi, and plants. The few exceptions include Glu–to-Gln transitions in Neurospora crassa and Dictostelium discoideum and Glu-to-Lys transitions in Apis mellifera, Plasmodium falciparum, Blastocystis hominis, and Tetrahymena thermophila.

DISCUSSION

In humans, mutations in the V-ATPase subunit genes B1 (ATP6V1B1), a3 (ATP6V0A3), and a4 (ATP6V0A4) are associated with distal renal tubular acidosis with sensorineural deafness (ATP6V1B1), distal renal tubular acidosis with variable sensorineural deafness (ATP6V0A4), or malignant infantile osteopetrosis (ATP6V0A3).^{7, 8, 23} A murine deletion model of the B1 subunit highlights the requirement of the B1 subunit in sustaining maximal urinary acidification.¹ Difficulties in studying human V-ATPase subunit mutations lie in the complexity of the multisubunit V-ATPase, the lack of clean cell culture models, and in the case of B1 subunit mutations, the ubiquitous presence of B subunits in mammalian cells. The yeast S. cerevisiae contains only one endogenous B subunit. Diploid null strains are readily available and have been thoroughly characterized.^{7, 16, 20} S. cerevisiae strains deficient in V-ATPase subunits are lethal secondary to a failure to lower the pH in the vacuolar system, and mutants only survive if a low external pH allows for vacuolar acidification presumably by fluid-phase endocytosis.⁷ Although mouse d and E subunits of the V-ATPase have been successfully used for functional complementation in yeast strains deficient in the cognate V-ATPase subunits, successful complementation with a mammalian V-ATPase B1 subunit has not been described.^{24, 25}

We demonstrate that the yeast S. cerevisiae is an ideal heterologous expression system for the functional analysis of human B1. The novel B1 subunit frameshift mutation (p.Phe468fsX487) illustrates the importance of the B1 subunit C-terminus in V-ATPase assembly. Furthermore, our analysis of all known B1 subunit missense mutations indicates lack of V₁ domain assembly as a rather common molecular mechanism of V-ATPase dysfunction in B1 subunit mutations. Further studies will have to be performed to address the exact mechanisms leading to disrupted V-ATPase assembly. In the case of p.Glu161Lys or p.Gly316Glu mutation, the reasons for loss of function despite intact assembly can be due to defective ATP binding and hydrolysis or uncoupling of ATP hydrolysis to H⁺ translocation.

Yang et al.⁹ demonstrated disrupted V-ATPase assembly in seven known ATP6V1B1 missense mutations using mammalian cells. Based on the fact that overexpressed mutant B1 subunits can lower Na⁺-independent cell pH recovery after acid load in a rat inner medullary collecting duct cell line, they proposed a dominant-negative mechanism of the transfected mutant B1 subunits over the native wild-type B1 subunit. In contrast, we did not find a dominant-negative effect of the novel frameshift mutation in our yeast assay. The discrepancy can be due to different expression system and different assays for H⁺-pump function. Dominant-negative effects have also been observed for the a3 and a4 subunits in a yeast complementation system.²⁶ The literature shows that heterozygously affected individuals of ATP6V1B1 missense mutations do not have a lower plasma bicarbonate concentration or higher urine pH but more provocative studies are not available. Thus, the notion of a dominant-negative effect raised by Yang et al.⁹ is highly intriguing and requires further investigation. Another interesting point is that the B2 isoform is expressed in mammalian cells and B2 has been shown to complement partially for loss of B1 in B1-null mice.^{27, 28, 29, 30}

For the p.Gly316Glu mutant B1 subunit, Yang et al.⁹ showed absence of interaction with the native E subunit. In our hands, the p.Gly316Glu mutant B1 subunit repeatedly co-immunoprecipitated with the native E subunit (Figure 5a) as well as subunits A and D (not shown), suggesting full V₁ assembly. The reasons for the divergent findings are currently unclear, but may lie in the different antibodies (-green fluorescent protein versus -FLAG) or epitope tagging (N-terminal GFP versus N-terminal FLAG) employed and require further testing.

Our finding of impaired function of a common ATP6V1B1 SNP (p.Glu161Lys) has important clinical implications, as it further extends the spectrum of V-ATPase-associated disease. This SNP was found to be in Hardy–Weinberg equilibrium in Turkish and Saudi Arabian control subjects with an estimated allele frequency of 10% .⁸ We currently do not have adequate phenotypic information on heterozygotes versus homozygotes with this variant allele. The frequency of this SNP will have to be determined in other populations and the phenotype defined by metabolic studies. A true allele frequency of 10% predicts homozygosity of 1:100, assuming strict adherence to simple Mendelian inheritance. It is conceivable that homozygously affected individuals may have impaired renal tubular acid secretion, especially under physiological stress situations. Partial impairment in distal tubular acid secretion is known as incomplete or partial distal renal tubular acidosis and has been associated with osteoporosis and nephrolithiasis.^{31, 32} These patients have normal blood pH and bicarbonate levels but fail to maximally acidify their urines when challenged. Systematic screening of high-risk populations may identify additional polymorphisms with reduced V-ATPase activity.

MATERIALS AND METHODS

Materials

Chemicals were obtained from Sigma-Aldrich (St Louis, MD, USA) except the following: Lipofectamine 2000 and restriction enzymes (Invitrogen, Carlsbad, CA, USA); PCR primers (Integrated DNA technologies, Coralville, IA, USA); Easy-A PCR mix (Stratagene, La Jolla, CA, USA); cell culture media and geneticin (Gibco BRL); fetal bovine serum (Atlanta Biologicals, Norcross, CA, USA); penicillin and streptomycin (Whittaker MA Bioproducts, Abbott Park, IL, USA); T4 DNA ligase and mini-EDTA-free protease inhibitor cocktail tablets (Roche); protein-G-agarose (Calbiochem, San Diego, CA, USA); rabbit polyclonal anti-FLAG, mouse monoclonal anti-FLAG, and HA antibodies (Sigma); horseradish peroxidase-conjugated sheep anti-mouse and donkey anti-rabbit IgG antibodies and enhanced chemiluminescence detection kit (Amersham, Beverly, MA, USA); polyvinylidene difluoride membranes (Immobilon, Billerica, MA, USA); Texas Red-conjugated goat anti-rabbit IgG antibody (Molecular Probes, Eugene, OR, USA); MDCK and HEK293 cells and S. cerevisiae YBR127C (Vma2p)-null strain (ATCC, Rockville, MD, USA); full-length Vma2p cDNA (Open Biosystems, Woburn, MA, USA); D-glucose (Fluka, St Louis, MD, USA); yeast nitrogen base (BD Biosciences, San Jose, CA, USA); -Ura DO supplement (Clontech, Palo Alto, CA, USA). Rabbit polyclonal antibody detecting V-ATPase E subunit was described earlier by Peng et al.³³

DNA analysis

The large Mexican family was characterized phenotypically.¹⁰ All homozygotes were affected but heterozygotes did not display clinically obvious metabolic acidosis or deafness. All participants gave informed consent to a protocol approved by the Institutional Review Board at UT Southwestern Medical Center. Genomic DNA was prepared from peripheral blood lymphocytes and ATP6V1B1 exons and exon–intron boundaries were amplified by PCR using fluorescently end-labeled primers on a PE Applied Biosystems model 377 automated DNA sequencer. The primers used have been described by Karet et al.^{7, 8} Samples were analyzed by 4% polyacrylamide gels and data analysis was performed using GENOTYPER software.

Mammalian and yeast expression constructs

Full-length human B1 subunit cDNA was amplified by PCR from a human kidney cDNA library (Clontech). Point mutations were introduced by PCR-based mutagenesis. cDNA fragments were inserted in-frame into C-terminal single HA-tagged PMH (Roche) or N-terminal triple FLAG-tagged p3xFLAG-CMV-10 (Sigma-Aldrich) vectors for mammalian expression constructs and into the p426TEF vector for expression in yeast.¹³ All yeast constructs contain a yeast translation initiation consensus sequence (AAAA) and two in-frame stop codons. C-terminal single HA-tags were introduced by PCR. All constructs were verified by sequencing.

Yeast transformation and growth selection

Expression constructs were transformed into S. cerevisiae (genotype MATa/MATalpha his31/his31 leu20/leu20 lys20/+ met150/+ ura30/ura30 VMA2/VMA2) and grown at 25°C in YEPD medium (10 g l^{- 1} yeast extract, 20 g l^{- 1} peptone, 20 g l^{- 1} D-glucose, 100 mg l^{- 1} adenine hemisulfate, 50 mM methanesulfonic acid, pH 5.5, or 50 mM 3-(N-morpholino)propanesulfonic acid, pH 7.5) or the synthetic minimal uracil (SD-Ura) dropout medium (-Ura DO supplement 0.77 g l^{- 1}, yeast nitrogen base 6.7, 20 g l^{- 1} D-glucose, 100 mg l^{- 1} adenine hemisulfate, 50 mM methanesulfonic acid, pH 5.5). All yeast media contained 200 g ml^{- 1} geneticin. Transformation of yeast was performed as described by Gietz et al.²¹ Single colonies were isolated on pH-5.5 SD-Ura plates and growth of transformants was assayed on YEPD plates buffered to pH 5.5 or 7.5.

For immunoblot, 10 o.d. units of yeast grown in SD-Ura medium were washed once with 1 mM EDTA/H2O and lysed in 2 M NaOH for 10 min on ice. Proteins were isolated by trichloroacetic acid/acetone precipitation and resuspended in SDS sample buffer (25 mM Tris-Cl, pH 6.8, 9 M urea, 1 mM EDTA, 1% (w/v) SDS, 0.7 M -mercaptoethanol, 10% (v/v) glycerol). After incubation (37°C, 15 min), equal portions of the lysates were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and analyzed by immunoblotting.

Cell culture and transfection

MDCK and HEK293 cells were cultured at 37°C in 95% air/5% CO₂ in high-glucose (450 mg dl^{- 1}) DMEM supplemented with 10% (v/v) fetal bovine serum, penicillin (100 U ml^{- 1}) and streptomycin (100 g ml^{- 1}). Transient transfections were performed with Lipofectamine 2000 and cells were used 36–48 h after transfection for further experiments.

Immunoprecipitation and immunoblotting

For immunoprecipitation, cells were grown in six-well plates and lysed in a buffer containing 0.5% (v/v) Triton X-100 in TBS with fresh protease inhibitors. The lysates were cleared by centrifugation (14 000 g 30 min), precipitating antibodies (1:200 dilution) and prewashed protein-G-agarose (30 l of 50% slurry) were added, and lysates were rotated for 4 h at 4°C. After washing three times with lysis buffer, proteins were eluted from the beads with 2.5 loading buffer (2.5 mM Tris-Cl, pH 6.8, 2.5% SDS, 2.5% -mercaptoethanol, 25% glycerol), heated (2 min, 95°C), separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Primary antibodies were used at a 1:1000 dilution, and secondary horseradish peroxidase-conjugated sheep anti-mouse and donkey anti-rabbit IgG antibodies were used at 1:20 000 dilutions, and signals were detected using enhanced chemiluminescence.

Immunocytochemistry

Forty hours after transfection, MDCK cells were treated with cylcoheximide (40 g ml^{- 1}) for 8 h. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked by 1.5% BSA/10% goat serum for 1 h. Specimens were incubated with rabbit anti-FLAG polyclonal antibody (1:200 dilution, 12 h, 4°C), followed by Texas Red-conjugated goat anti-rabbit IgG antibody (1:500 dilution; 2 h). Confocal fluorescence images were acquired through a Zeiss 100 objective lens using a Zeiss LSM-510 laser-scanning confocal microscope. Fluorescence of Texas Red and DAPI were detected using excitation laser wavelengths (in nm) of 568 and 350, respectively.

Equipment and settings

Immunoblots were scanned with ScanMaker X6EL (Microtek, Cerritos, CA, USA) scanner and Adobe Photoshop (version 7.0) software (resolution of 600 dpi) and otherwise default settings. Blot images were cropped, combined in CorelDraw (version 11), and then converted to TIFF files without image processing. Images of yeast plates were acquired using a Canon S2 IS digital camera at 5-megapixel resolution as low-compression JPEG files, and without further processing, images were cropped and converted to grayscale TIFF files. Individual experiments were repeated at least three times.

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Acknowledgments

We thank Martha Lemke for technical assistance, Steve Gluck and Kiyoshi Ariizumi for helpful advice, and Albert Quan and Henry Quiñones for patient referral. This work was supported by National Institutes of Health Grants DK-48482 and DK-20543 to O.W.M. D.G.F. was partially supported by a stipend from the Swiss National Science Foundation and a Fellowship from the Charles and Jane Pak Center of Mineral Metabolism and Clinical Research.