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. 1998 Oct;18(10):5952–5960. doi: 10.1128/mcb.18.10.5952

Functional Activity of the Fanconi Anemia Protein FAA Requires FAC Binding and Nuclear Localization

Dieter Näf 1, Gary M Kupfer 1,2, Ahmed Suliman 1, Kathleen Lambert 1, Alan D D’Andrea 1,2,*
PMCID: PMC109181  PMID: 9742112

Abstract

Fanconi anemia (FA) is an autosomal recessive disease characterized by genomic instability, cancer susceptibility, and cellular hypersensitivity to DNA-cross-linking agents. Eight complementation groups of FA (FA-A through FA-H) have been identified. Two FA genes, corresponding to complementation groups FA-A and FA-C, have been cloned, but the functions of the encoded FAA and FAC proteins remain unknown. We have recently demonstrated that FAA and FAC interact to form a nuclear complex. In this study, we have analyzed a series of mutant forms of the FAA protein with respect to functional activity, FAC binding, and nuclear localization. Mutation or deletion of the amino-terminal nuclear localization signal (NLS) of FAA results in loss of functional activity, loss of FAC binding, and cytoplasmic retention of FAA. Replacement of the NLS sequence with a heterologous NLS sequence, derived from the simian virus 40 T antigen, results in nuclear localization but does not rescue functional activity or FAC binding. Nuclear localization of the FAA protein is therefore necessary but not sufficient for FAA function. Mutant forms of FAA which fail to bind to FAC also fail to promote the nuclear accumulation of FAC. In addition, wild-type FAC promotes the accumulation of wild-type FAA in the nucleus. Our results suggest that FAA and FAC perform a concerted function in the cell nucleus, required for the maintenance of chromosomal stability.

Fanconi anemia (FA) is an autosomal recessive disease characterized by genomic instability, cancer susceptibility, progressive bone marrow failure, and selective cellular hypersensitivity to bifunctional alkylating agents (1, 5, 10). Somatic cell fusion studies have defined eight complementation groups of FA (FA-A through FA-H), suggesting the possibility of as many as eight FA genes (3, 14, 15). The genes corresponding to FA-A and FA-C have been cloned (12, 23, 33), and mutations in FAA and FAC account for approximately 80% of FA patients (3, 15). The FAA and FAC proteins have no sequence similarity to each other or to other proteins in GenBank, and their biochemical functions remain unknown.

Cells derived from FA patients display multiple phenotypic abnormalities (10, 22). FA cells are hypersensitive to bifunctional alkylating agents such as diepoxybutane and mitomycin C (MMC), suggesting a defect in DNA repair. FA cells also exhibit abnormal cell cycle progression (16, 18, 19) and reduced cell survival (6, 9, 24, 28, 29, 35). Many of these abnormalities are also evident in primary cells derived from mice homozygous for a disrupted fac gene (8, 35). How the FA proteins regulate these cellular activities remains unknown.

FAA and FAC have recently been shown to physically interact and form a nuclear complex (20). A mutant form of FAC (FACL554P), expressed in a patient-derived FA-C cell line, failed to bind FAA, suggesting that the biological function of the FA proteins requires formation of an FAA-FAC complex. Since this protein complex is found in the nuclei of normal cells, the FA proteins presumably mediate some nuclear function, perhaps related to DNA repair, transcription, or RNA processing.

Little is known regarding the nature of the binding interactions between the FAA and FAC proteins. For instance, the binding may be a direct protein-protein interaction or may be an indirect interaction, mediated by other adaptor proteins. Regulated posttranslational modification of the FAA or FAC protein, such as phosphorylation, may also be required for interaction of the FA proteins.

To assess the functional importance of FAC binding and nuclear localization, we expressed mutant forms of FAA in the MMC-sensitive FA-A fibroblast cell line GM6914. The mutant FAA proteins were analyzed with respect to correction of cellular MMC sensitivity, FAA-FAC interaction, and nuclear localization of the FA protein complex. Our results demonstrate that FAC binding and nuclear localization are both required for the biological function of the FAA protein. Moreover, FAC binding is required for the nuclear uptake and accumulation of the FAA protein.

MATERIALS AND METHODS

Plasmid constructs.

Wild-type and mutant FAA cDNAs were subcloned into the retroviral expression vector pMMP (26) according to standard procedures (2). Mutations in the FAA cDNA were introduced by PCR with Pfu polymerase (Stratagene). The FAAΔXho cDNA was generated by deletion of an internal XhoI restriction fragment (2,105 bp) from the FAA open reading frame. The cDNA inserts were verified by DNA sequencing.

Cell culture.

GM6914 fibroblasts (American Type Culture Collection) were maintained in Dulbecco modified Eagle medium (DMEM) containing 15% (vol/vol) fetal calf serum (FCS). Cells were grown at 37°C in a humidified atmosphere containing 5% CO2. 293GPG producer cells (26) were cultivated in DMEM–10% FCS supplemented with tetracycline (1 μg/ml; Sigma), Geneticin (0.3 mg/ml; Gibco), and puromycin (2 μg/ml; Sigma). Lymphoblasts derived from a normal adult (PD7), an FA-A patient (HSC72), and an FA-C patient (HSC536) have previously been described (33, 36).

Production of pMMP-FAA retroviral supernatants and infection of GM6914 cells.

For production of pMMP-FAA retrovirus, 293GPG helper cells (26) were grown to 90% confluency in 10-cm-diameter dishes and transfected for 8 h at 37°C with plasmid DNA (10 μg) in 6 ml of Opti-MEM (Gibco) containing 8.7 μl of Lipofectamine (Gibco) per ml. The medium was replaced with DMEM–10% FCS (10 ml) and changed every 24 h. Virus-containing supernatants were harvested 96, 120, and 144 h after lipofection and clarified by filtration (0.45-μm-pore-size filter). Viral supernatants (5 ml) were pooled and mixed with an equal volume of DMEM–15% FCS containing 8 μg of Polybrene (hexdimethrine bromide; Sigma) per ml. The mixture was added to 10-cm-diameter dishes containing GM6914 cells (approximately 2 × 105) seeded the previous day. After incubation for 4 to 6 h at 37°C in 5% CO2, the medium was replaced with DMEM–15% FCS. Infection efficiencies ranged from 60 to 80%, as estimated by 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) staining of plates infected in parallel with pMMP-nlsLacZ (26).

MMC sensitivity assay.

Retrovirus-infected GM6914 cells were seeded onto six-well plates at 1.5 × 104 cells/well in DMEM–15% FCS (5 ml). After cells attached for 16 to 24 h, the medium was replaced with DMEM–15% FCS containing MMC (Aldrich) at various concentrations. After incubation for 9 to 10 days, monolayers were washed twice with phosphate-buffered saline (PBS) and fixed for 5 to 10 min at 23°C in 10% (vol/vol) methanol and 10% (vol/vol) acetic acid. Adherent colonies were stained for 2 to 10 min at 23°C with 1% (wt/vol) crystal violet (Sigma) in methanol (0.5 ml per well). Plates were rinsed in distilled water, and the adsorbed dye was resolubilized with methanol containing 0.1% (wt/vol) sodium dodecyl sulfate SDS (0.5 ml per well) by gentle agitation for 1 to 4 h at 23°C. Dye solution (150 μl) was transferred to 96-well plates and diluted (1:3) in methanol. Crystal violet concentrations were measured photometrically (595 nm) in a model 3550 microplate reader (Bio-Rad). For quantitation, readings of optical density at 595 nm were normalized to those obtained from untreated cells (concentration of MMC = 0 nM), assumed to yield 100% cell survival.

Western blotting.

Western blotting and immunoprecipitation of FAA were performed as described previously, using affinity-purified polyclonal rabbit antisera (20). The anti-FAA(N) and anti-FAA(C) antisera were raised against the N and C termini, respectively, of FAA (20).

Immunofluorescence microscopy.

Cells were seeded onto four-well chamber slides (Falcon) and cultivated for 16 to 24 h. Slides were rinsed with PBS, and adherent cells were fixed for 20 min at 23°C in paraformaldehyde (4% [wt/vol] in PBS) and permeabilized with Triton X-100 (0.3% [vol/vol] in PBS) for 10 min at 23°C. Staining with primary [affinity-purified anti-FAA(C)] and secondary (fluorescein-conjugated goat anti-rabbit) antibodies was for 2 h at 23°C, followed by counterstaining for 5 min at 23°C with DAPI (4′,6-diamidino-2-phenylindole dihydrochloride; 10 μg/ml in PBS; Sigma). Slides were mounted in Vectashield (Vector Laboratories) and analyzed by fluorescence microscopy.

Cell fractionation.

Fractionation of GM6914 cells into nuclear and cytoplasmic fractions was performed as previously described (20). For fractionation of lymphoblasts, cells were washed in PBS, then resuspended in buffer (10 mM Tris [pH 7.4], 3 mM CaCl2, 2 mM MgCl2, 1% Nonidet P-40), and fractionated in a Dounce homogenizer with 30 strokes. Nuclei were pelleted by centrifugation at 1,500 rpm, and the supernatant (cytosolic fraction) was clarified by centrifugation at 15,000 rpm. Nuclei were washed three times with PBS and lysed in buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Triton X-100). Nuclear lysates were clarified by centrifugation at 15,000 rpm in a microcentrifuge.

RESULTS

Generation of retroviral vectors encoding mutant FAA proteins.

The wild-type FAA cDNA encodes a polypeptide of 1,455 amino acids with a putative bipartite nuclear localization signal (NLS) at its amino terminus (Fig. 1A). Initially, FAA cDNAs encoding various mutant FAA polypeptides were generated. The FAAΔNLS protein lacks the first 36 amino acids of FAA, including the putative NLS. The SV40T-FAA protein contains the 13-amino-acid NLS of the simian virus 40 (SV40) T antigen (SV40T) replacing the putative NLS of FAA. FAAΔXho is a truncated protein with an intact amino terminus but lacking 853 carboxyl-terminal amino acids.

FIG. 1.

FIG. 1

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Schematic representation of wild-type FAA and mutant proteins. (A) The wild-type FAA protein is 1,455 amino acids in size and contains a bipartite NLS (black bar) and a partial leucine zipper (Leu Zip; gray bar) motif. cDNAs encoding several mutant FAA proteins were generated as described in the text. The FAAΔXho polypeptide is truncated at amino acid 592 and contains 17 additional amino acids at its carboxyl terminus. (B) The specific amino acid changes in the N termini of mutant FAA polypeptides are shown. The basic amino acids of the bipartite NLS are highlighted by shading.

In addition, we generated several FAA cDNAs with missense mutations in the 5′ region of the open reading frame (Fig. 1B). The FAA-NLS-mut1 protein contains point mutations in the carboxyl half of the bipartite NLS sequence, and all basic amino acids of the NLS were mutated in FAA-NLS-mut2.

The FAA-V6D and FAA-N8K variants were generated, based on the identification of these presumably benign polymorphisms found in FA-A families (21). While there is no evidence that these mutations are pathogenic in FA-A patients, the construction of these mutant cDNAs allows the functional assessment of these variant FA-A proteins.

Wild-type and mutant FAA cDNAs were inserted into the retroviral expression plasmid pMMP and transfected into the 293GPG producer cell line (26). Supernatants containing high titers of pMMP-FAA retroviruses were used to infect GM6914 (FA-A) fibroblasts, an MMC-sensitive cell line with no detectable endogenous FAA protein.

The amino terminus of FAA is required for functional complementation of FA-A cells.

Retrovirally transduced GM6914 cells were initially analyzed for MMC sensitivity (Fig. 2) and FAA protein expression (Fig. 3). Consistent with previous reports (20), GM6914 cells infected with pMMP-FAA(wt) were resistant to MMC (Fig. 2A). Cells infected with pMMP-nlsLacZ retained their MMC sensitivity, indicating that retroviral infection alone did not alter the GM6914 cellular phenotype. The abilities of the various mutant forms of FAA to complement MMC-sensitive GM6914 cells are summarized in Fig. 2B.

FIG. 2.

FIG. 2

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Amino-terminal and carboxyl-terminal regions of FAA are required for the biochemical function of FAA. Following retroviral transduction, the indicated FAA polypeptides were expressed in the FA-A fibroblast cell line GM6914. Infected cells were grown in the presence of various concentrations of MMC, and cell survival was assayed by crystal violet staining of viable colonies. (A) Representative experiment showing sensitivity of retrovirally transduced GM6914 cells across several MMC concentrations. At 15 nM MMC, corrected GM6914 cells showed enhanced survival compared to uncorrected GM6914 cells (60 and 0% survival, respectively) (B) Sensitivity of infected GM6914 cells in 15 nM MMC. Bars represent averages and standard deviations of six independent experiments.

FIG. 3.

FIG. 3

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Mutant FAA polypeptides have differential binding activity for the FAC polypeptide. FA-A fibroblasts (GM6914), expressing wild-type (wt) or mutant forms of the FAA polypeptide, were analyzed for expression and binding of FAA and FAC. Protein from the indicated cell lines was immunoprecipitated (IP) with a 1:1 mixture of anti-FAA(C) and anti-FAA(N) antisera. Protein was electrophoresed, transferred to nitrocellulose, and immunoblotted with either anti-FAA(N) or anti-FAC(C) antiserum. A whole-cell extract (WCE) from each cell line was also analyzed in parallel to assess the expression of endogenous FAC protein (bottom panel). An arrowhead indicates the immunoglobulin heavy chain. As previously described, the FAC protein in GM6914 fibroblasts is expressed as multiple isoforms, shown as two bands in most of the anti-FAC immunoblots. Cells were infected with either pMMP-nlsLacZ (lanes 1 and 14), pMMP-FAA(wt) (lanes 2, 5, and 10), pMMP-FAAΔNLS (lanes 4, 7, and 11), pMMP-FAA-NLS-mut1 (lanes 3 and 8), pMMP-FAA-NLS-mut2 (lane 9), pMMP-FAAΔXho (lane 6), pMMP-FAA-V6D (lane 12), or pMMP-SV40T-FAA (lane 13).

The FAAΔNLS, FAAΔXho, and SV40T-FAA proteins had decreased activity in the MMC assay, indicating that both N- and C-terminal domains of FAA are required for full biological activity of the FAA protein. Ablation of the basic amino acids in both sections of the bipartite NLS region (FAA-NLS-mut2) abolished complementation activity. In contrast, the FAA-NLS-mut1, FAA-V6D, and FAA-N8K proteins demonstrated normal function in the MMC sensitivity assay. Taken together, these results indicate that the amino-terminal bipartite NLS motif and the carboxyl terminus of FAA are critical for FAA function in vitro. Replacement of the amino terminus of FAA with the heterologous NLS from SV40T did not rescue the biological function of FAA.

The amino terminus of FAA is required for interaction with FAC.

GM6914 cells infected with the various pMMP-FAA constructs were next assayed for expression of the virally encoded polypeptides and for FAA-FAC binding (Fig. 3). All variant FAA proteins were expressed at similar levels. As predicted by its cDNA sequence, the FAAΔNLS polypeptide was slightly smaller (approximately 155 kDa) (lanes 4, 7, and 11) than the wild-type FAA protein (163 kDa) (lanes 2, 5, and 10). Also, the electrophoretic mobility of the FAAΔXho polypeptide matched its predicted molecular mass (63 kDa) (lane 6). Missense mutants of FAA had the same electrophoretic mobility as the wild-type protein. No endogenous FAA protein was detectable in parental GM6914 cells or in cells infected with negative control viruses (20) (lanes 1 and 14).

To test the mutant FAA proteins for their interaction with endogenous wild-type FAC, we analyzed anti-FAA immune complexes for the presence of FAC by Western blotting with an antiserum specific to the carboxyl terminus of FAC (36). FAC coimmunoprecipitated with wild-type FAA (Fig. 3, FAC immunoblot, lanes 2, 5, and 10). Interestingly, all functionally deficient FAA mutants tested, including FAAΔXho (lane 6), FAAΔNLS (lanes 4, 7, and 11), FAA-NLS-mut2 (lane 9), and SV40T-FAA (lane 13), failed or weakly coimmunoprecipitated with FAC, indicating that the biological activity of FAA correlated with its ability to bind to FAC. Overexpression of retrovirally encoded FAA polypeptides did not affect steady-state levels of endogenous (wild-type) FAC (Fig. 3, anti-FAC immunoblot of whole-cell extracts).

Nuclear localization of FAA is necessary but not sufficient for functional complementation.

We next analyzed the subcellular localization of the various mutant FAA polypeptides by immunofluorescence microscopy, using an affinity-purified anti-FAA(C) antiserum (Fig. 4). Cells expressing wild-type FAA protein displayed FAA-specific staining predominantly in the nucleus, with a faint diffuse staining of the cytoplasm. A few cells expressing the wild-type FAA protein also showed a speckled fluorescent nuclear pattern (data not shown), suggesting that FAA may accumulate in a critical subnuclear compartment. In contrast, predominantly cytoplasmic staining was observed in GM6914 cells expressing FAAΔNLS or FAA-NLS-mut2, indicating that deletion or mutation of both sections of the bipartite NLS motif diminished nuclear localization. Interestingly, the SV40T-FAA protein displayed nuclear localization, although it did not functionally complement the GM6914 cells (Fig. 2) or bind FAC (Fig. 3).

FIG. 4.

FIG. 4

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Nuclear localization of wild-type FAA and SV40T-FAA. The FA-A fibroblast line GM6914 does not express detectable FAA protein. GM6914 cells were infected with pMMP-FAA(wt), pMMP-FAAΔNLS, pMMP-FAA-NLS-mut2, pMMP-SV40T-FAA, or pMMP-nlsLacZ (encoding β-galactosidase). Cells infected with pMMP-FAA(wt) were corrected to MMC resistance, while cells infected with pMMP-FAAΔNLS, pMMP-FAA-NLS-mut2, or pMMP-nlsLacZ remained MMC sensitive (Fig. 2). Pools of infected cells were stained with anti-FAA(C) and the DNA-specific dye DAPI and analyzed by immunofluorescence as described in the text.

Since the anti-FAA(C) antiserum did not react with the C-terminally truncated FAAΔXho polypeptide, which is missing the C-terminal epitope recognized by the antibody, we used a cell fractionation strategy (20) to assess the cellular distribution of FAAΔXho (Fig. 5). The FAAΔXho protein was found predominantly in cytoplasmic extracts prepared from infected GM6914 cells (lane 6), with only minute amounts detected in nuclear fractions (lane 9). In contrast, full-length FAA was found in both the nuclear and cytosolic fractions of cells expressing the wild-type protein (lanes 5 and 8), in agreement with the immunofluorescence results (Fig. 4). The amount of nuclear FAC protein was significantly higher in GM6914 cells expressing wild-type FAA (anti-FAC immunoblot, lane 8) than in cells containing FAAΔXho (anti-FAC immunoblot, lane 9), suggesting that C-terminal sequences of FAA are required for nuclear accumulation of the FAA-FAC complex.

FIG. 5.

FIG. 5

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The carboxyl terminus of FAA is required for FAC binding and nuclear localization of the FAA-FAC complex. GM6914 fibroblasts infected with either pMMP-nlsLacZ (lanes 1, 4, and 7), pMMP-FAA(wt) (lanes 2, 5, and 8), or pMMP-FAAΔXho (lanes 3, 6, and 9) were fractionated into cytoplasmic and nuclear extracts as previously described (20). Total and fractionated cell extracts were analyzed by immunoblotting with either anti-FAA(N) or anti-FAC(C) antiserum. The fractions were also analyzed by antitubulin immunoblotting to ensure adequate cellular fractionation. Sizes (in kilodaltons) are indicated on the right.

To confirm the requirement of FAA-FAC binding in nuclear localization of FAC, we fractionated GM6914 fibroblasts expressing various mutant forms of FAA (Fig. 6). Wild-type and mutant FAA proteins were expressed at relatively equal levels in the cells. All cell lines expressed similar amounts of FAC protein (total FAC immunoblot). Cells expressing wild-type FAA protein or FAA-NLS-mut1 demonstrated higher levels of FAC protein in the nuclear fraction (nuclear FAC immunoblot, lanes 2 and 4, respectively). In contrast, GM6914 cells expressing no FAA protein (lane 1) or mutant forms of FAA which fail to bind FAC (lanes 3 and 5) had only little FAC protein in the nuclear fraction. By a similar analysis, the SV40T-FAA fusion protein, which failed to bind to FAC (Fig. 3), also failed to promote FAC accumulation in the nucleus (data not shown).

FIG. 6.

FIG. 6

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Nonfunctional mutant forms of the FAA protein fail to promote FAC accumulation in the nucleus. GM6914 fibroblasts infected with either pMMP-nlsLacZ (lanes 1), pMMP-FAA(wt) (lanes 2), pMMP-FAAΔNLS (lanes 3), pMMP-FAA-NLS-mut1 (lanes 4), or pMMP-FAA-NLS-mut2 (lanes 5) were fractionated into cytoplasmic and nuclear extracts. The indicated fractions were analyzed by immunoblotting with either anti-FAA(N) or anti-FAC(C) antiserum (36). Fractions were also analyzed by anti-β-tubulin immunoblotting to ensure adequate cellular fractionation.

FAA-FAC binding is required for nuclear localization and accumulation of the protein complex.

To confirm the nuclear localization of wild-type FAA protein observed in fibroblasts, we next analyzed various human lymphoblast lines (Table 1). The FA-A lymphoblast line HSC72 was sensitive to MMC, with a 50% effective concentration (EC50) of 13 nM MMC. Stable transfection of these cells with plasmid pREP4-FAA resulted in functional complementation and an EC50 of 118 nM MMC. Analogously, transfection of the FA-C cell line HSC536N with plasmid pREP4-FAC corrected its MMC sensitivity. We have previously shown that the FAA and FAC proteins coimmunoprecipitate from extracts prepared from functionally complemented HSC72/FAA(wt) cells or HSC536N/FAC(wt) cells (20).

TABLE 1.

Characterization of FA lymphoblast lines

Cell line/plasmid Complementation group Mean MMC EC50 (nM) ± SE FAA-FAC bindinga
HSC72 FA-A 13 ± 5 −
HSC72/FAA(wt) FA-A 118 ± 16 +
HSC536N FA-C 14 ± 5 −
HSC536N/FAC(wt) FA-C 121 ± 14 +
PD7 Wild type 145 ± 23 +
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a

From reference 20. 

We next tested the localization of FAA and FAC in these lymphoblast lines by cell fractionation (Fig. 7). The FA-A line, HSC72, expressed no detectable endogenous FAA protein (FAA immunoblot, lanes 2, 7, and 12), as previously described (20). Low levels of FAC were observed in the nuclei of these cells (FAC immunoblot, lane 12), although most FAC was detected in the cytoplasm, consistent with previous studies (13, 36, 38). In contrast, HSC72 cells corrected with FAA cDNA expressed FAA protein (FAA immunoblot, lanes 3, 8, and 13). In these cells, FAA was found in similar amounts in the cytoplasm and nucleus (compare lanes 8 and 13). Increased levels of the FAC protein were observed in the nuclei of the corrected cells (FAC immunoblot, lane 13). These results demonstrate that FAA is required for efficient FAC localization and accumulation in the nucleus.

FIG. 7.

FIG. 7

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Expression of wild-type FAC is required for nuclear accumulation of wild-type FAA. The indicated lymphoblast lines were fractionated into total, cytoplasmic, and nuclear extracts as previously described (20). Fractions were analyzed by immunoblot analysis with either anti-FAA(N), antiserum anti-FAC(C) antiserum, or anti-tubulin antibody. Cell lines examined were PD7 (lanes 1, 6, and 11), HSC72 (lanes 2, 7, and 12), HSC72 corrected with wild-type FAA (lanes 3, 8, and 13), HSC536N (lanes 4, 9, 14), and HSC536N cells corrected with wild-type FAC (lanes 5, 10, and 15). An amino-terminally truncated isoform of FAC, called FRP-50 (36), localized in the cytoplasm but not nuclear fractions, as previously described (20). Tubulin was excluded from nuclear fractions, ensuring proper cell fractionation.

In the uncorrected FA-C cell line, HSC536N, wild-type FAA protein was localized primarily in the cytoplasm (FAA immunoblot, lane 9 versus lane 14). Following stable transfection with an FAC expression plasmid, the corrected HSC536N cells expressed wild-type FAC protein, and the FAA protein localized predominantly to the nucleus (FAA immunoblot, lane 15). In these cells, increased FAC was also observed in the nucleus (FAC immunoblot, lane 15). Taken together, these results demonstrate that FAA and FAC binding is required for the normal nuclear accumulation of the protein complex. Each protein depends on the other for efficient nuclear localization.

DISCUSSION

GM6914, an immortalized fibroblast cell line derived from a patient of complementation group FA-A, is well suited for structure-function studies of FAA. These cells are sensitive to MMC, express normal levels of wild-type FAC protein, and contain no detectable FAA protein. Wild-type and mutant FAA cDNAs can be introduced into these cells by retroviral gene transfer with high efficiency, leading to stable expression of the proteins. Infection of GM6914 cells with a pMMP-derived retroviral vector carrying the wild-type FAA cDNA leads to functional complementation of MMC hypersensitivity, expression of the FAA polypeptide, formation of the FAA-FAC protein complex, and nuclear accumulation of FAA and FAC. In this study, we have exploited this cellular system and analyzed the structural features of FAA required for its functional properties.

Based on our series of mutant FAA polypeptides, we conclude that the biological function of the FAA protein depends on its ability to bind to FAC and to accumulate in the nucleus (summarized in Table 2). Mutant forms of the FAA protein, such as FAAΔNLS, FAA-NLS-mut2, and FAAΔXho, which fail to bind FAC and fail to accumulate in the nucleus, are biologically inactive. Interestingly, SV40T-FAA, which does translocate to the nucleus, fails to bind FAC and is nonfunctional.

TABLE 2.

Functional analysis of FAA proteins

Protein Complementation of FA-A cells Binding to FAC Nuclear localization
Wild-type FAA + + +
FAAΔNLS − − −
SV40T-FAA − − +
FAA-NLS-mut1 + + +
FAA-NLS-mut2 − − −
FAAΔXho − − −
FAA-V6D + + +
FAA-N8K + + +
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Our data clearly demonstrate that the amino terminus of FAA is required for nuclear localization, FAC binding, and functional activity. According to several criteria, the amino terminus of FAA appears to contain a bona fide bipartite NLS sequence. Deletion of this region or mutation of both portions of the NLS ablates nuclear localization. Consistent with previous studies (30), mutation of one portion of the bipartite NLS sequence does not affect functional activity. Replacement of the bipartite NLS with the 13-amino-acid NLS of SV40T rescues nuclear localization. Presumably the NLS of FAA mediates nuclear uptake through its direct interaction with general nuclear translocation proteins, such as importin-α and importin-β (7, 11, 27).

How the amino terminus of the FAA protein mediates FAC binding is less clear. Several models are plausible. First, FAA and FAC may bind directly, with the N terminus of FAA serving as an FAC binding domain. Second, FAA and FAC may bind directly, but only after the regulated posttranslational modification of FAA and/or FAC. The amino terminus of FAA may be required for such a modification. Third, FAA and FAC may bind indirectly, through other proteins in the complex. Some of these putative adaptor proteins may be encoded by other FA genes. Recent studies suggest a requirement for adaptor proteins or posttranslational modifications in FAA-FAC binding. For instance, in vitro-translated FAA and FAC proteins fail to bind directly in the absence of cellular extracts (12a). Also, the nuclear FAA-FAC protein complex has a very high molecular mass, consistent with the presence of additional (unknown) protein subunits of the complex (18a).

The functional requirement for FAA-FAC binding has several possible mechanistic interpretations. FAC binding may be required to stabilize or retain FAA in the nucleus or to recruit additional proteins to the nuclear complex. Alternatively, FAC binding may be required to prevent the dephosphorylation of FAA. Consistent with this latter model, FAA is phosphorylated in cells expressing wild-type FAC but unphosphorylated in cells with mutant FAC which fails to bind FAA (37).

The failure of the SV40T-FAA protein to complement GM6914 cells is particularly interesting and has several explanations. The most likely explanation is that the N-terminal NLS region of FAA overlaps with or contributes to the FAC binding site. Substitution of this region with the NLS of SV40T allows nuclear uptake but ablates FAC binding. Accordingly, the FAC-FAA complex cannot form and accumulate in the nucleus. Second, the SV40T NLS sequence may function as a constitutive NLS which rapidly drives proteins directly to the nucleus, via the importin-α/importin-β pathway. In contrast, the NLS sequence of FAA may be a conditional NLS sequence, which requires additional protein interactions or posttranslational modification before it becomes active. Such examples of conditional, regulated NLS sequences have previously been described (25). Third, removal of the N-terminal 36 amino acids of FAA might ablate its interaction with some other protein, thereby disrupting FAA activity. Finally, it is possible that the SV40T-FAA fusion protein fails to translocate to a critical subcompartment of the nucleus where wild-type FAA normally accumulates. An understanding of the molecular basis for the difference between wild-type FAA and the SV40T-FAA will require further study.

While our data demonstrate the importance of the amino terminus of FAA in nuclear uptake, FAC binding, and functional activity, our study has several limitations. First, it is important to recognize that the accumulation of FAA and FAC in the nucleus, as measured by immunofluorescence or cell fractionation, is a steady-state measurement and depends on the individual rates of nuclear import, nuclear export, and protein degradation in the nucleus. The extent to which the amino terminus of FAA affects these individual rates has not been determined.

Second, our study suggests, but does not prove, the existence of C-terminal sequences of FAA required for FAA activity. For instance, the FAAΔXho protein, which is missing the C terminus of FAA, is expressed weakly in the nuclei of retrovirally infected cells (Fig. 5) but does not accumulate to levels observed for full-length FAA protein. Since FAAΔXho does not bind FAC in our coimmunoprecipitation assay (Fig. 3), FAC binding does not appear to be absolutely required for nuclear import of FAA but instead may be required for stabilization of FAA in the nucleus. Consistent with these results for the FAAΔXho mutant, we have generated a fusion of the amino-terminal 100 amino acids of FAA, including the NLS, with the green fluorescence reporter protein (FAA-NLS-GFP) (data not shown). Expression of FAA-NLS-GFP in GM6914 cells results in diffuse cytoplasmic and nuclear staining, without a distinct nuclear accumulation of the signal. Taken together, these studies suggest that C-terminal sequences of the FAA protein might also affect nuclear accumulation. Since the FAAΔXho mutant and FAA-NLS-GFP have large C-terminal deletions of FAA, smaller in-frame deletions or point mutations will be required to further assess the functional importance of the C terminus.

Our data conflict with a recent study by Kruyt et al. (17), suggesting that FAA is a cytoplasmic protein. A reason for these discrepancies may derive from the use of different cell lines in the previous report (17). For instance, Kruyt et al. used FAA-overexpressing 293 cells for FAA localization studies and HSC72 cells for FAA functional analysis, whereas we used functionally complemented FA-A cells and an antiserum to FAA in order to assess localization directly in these cells. Our studies are consistent with those of Hoatlin et al. (13), which demonstrate FAC in the cytoplasm and the nucleus and an increase in nuclear FAC in HSC536N cells following correction with wild-type FAC. Our data also demonstrate that FAA accumulation in the nucleus requires a functional NLS at the amino terminus.

Recent studies have demonstrated many different mutant and polymorphic FAA alleles (12, 21, 23). Our expression studies help to distinguish true mutant FAA polypeptides from polymorphic variants with functional activity. For instance, the FAA-V6D and FAA-N8K variant proteins appear to have normal functional activity in vitro, with respect to biological function, FAC binding, and nuclear localization (Table 2), consistent with their classification as polymorphisms rather than true mutations (21). Whether these variant proteins have differential activity in vivo remains to be tested.

Finally, the identification of additional components of the FAA-FAC complex may help to define the biochemical function(s) of the FA proteins. For instance, recent studies have shown that the tumor suppressor protein BRCA1 interacts with the DNA repair protein Rad51 (31, 32, 39). This interaction provided new insight to the molecular function of BRCA1 and underscored the importance of DNA repair processes in the maintenance of genomic integrity. Other protein complexes, such as the Rad50-Mre11-nibrin complex, are defective in known chromosome instability syndromes (4, 34). It remains to be determined whether the FA proteins similarly interact with known DNA repair proteins in the nucleus.

ACKNOWLEDGMENTS

We thank M. Grompe and H. Joenje for FA cell lines and R. Mulligan for the 293GPG packaging cells and the pMMP vector.

This work was supported by NIH grants R01-H15725 and PO1CA39542. G.M.K. is supported by grant K08-H103420. D.N. is a Fellow of the Leukemia Society of America (LSA), and A.D.D. is a Scholar of the LSA.

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