©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Cytosolic Proteins That Bind to the Fanconi Anemia Complementation Group C Polypeptide in Vitro
EVIDENCE FOR A MULTIMERIC COMPLEX (*)

Hagop Youssoufian (1)(§), Arleen D. Auerbach (2), Peter C. Verlander (2), Viktor Steimle (3), Bernard Mach (3)

From the (1) Department of Medicine, Hematology-Oncology Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, the (2) Laboratory of Investigative Dermatology, The Rockefeller University, New York, New York 10021, and the (3) Department of Genetics and Microbiology, University of Geneva Medical School, CH-1211 Geneva 4, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The oligomeric structure of Fanconi anemia complementation group C (FACC) was investigated in mammalian cell lysates. Using an affinity-purified polyclonal antibody, FACC was immunoprecipitated from radiolabeled cell lysates and shown to form monomers of 63 kDa. Association of FACC with heterologous proteins was investigated by co-precipitation of radiolabeled proteins with a recombinant chimeric FACC molecule fused to the constant portion of the human IgG1 heavy chain (FACC1). Expression of FACC1 in FACC-deficient Fanconi anemia (FA) lymphoblasts corrected the hypersensitivity of these cells to mitomycin C. Binding of FACC1 to protein A-agarose and incubation with radiolabeled cell lysates identified three polypeptides with molecular masses of 65, 50, and 35 kDa that were also detected on immunoblots probed with the purified FACC1 polypeptide. FACC, as well as the three FACC-binding polypeptides, co-fractionated with cytosolic and membrane extracts. Binding was specific for the FACC moiety of FACC1 and was detected in cytosolic extracts of a number of FA and non-FA mammalian cells. These results demonstrate that FACC binds directly to a family of ubiquitous cytosolic proteins and is conserved in a wide range of mammalian cells.


INTRODUCTION

Fanconi anemia (FA)() is an autosomal recessive disorder characterized by congenital malformations, bone marrow failure, the development of malignant disorders, and cellular hypersensitivity to DNA bifunctional cross-linking agents (1, 2, 3, 4, 5) , such as diepoxybutane and mitomycin C (MMC). Although these features have often led to the classification of FA with other disorders of DNA repair, the precise biochemical defects have not yet been elucidated.

Somatic cell fusion studies have revealed at least four complementation groups of FA (6, 7) . The gene for complementation group C was isolated by virtue of its ability to correct the hypersensitivity of FA group C (FA-C) cells to DNA bifunctional cross-linking agents (8) . The subsequent identification of mutations in this gene (8, 9, 10, 11, 12) established its unequivocal importance in the pathobiology of FA and provided an important reagent in the pursuit of the fundamental biochemical defects of FA. The full-length cDNA encodes a polypeptide of 558 amino acids (8, 13) with a predicted molecular mass of 63 kDa. Although no significant homologies have emerged from a comparison of the FACC sequence with those of other proteins in data bases, the predicted polypeptide sequence contains a preponderance of hydrophobic residues, as well as consensus sites for Ser/Thr phosphorylation and for binding to the molecular chaperone immunoglobulin heavy chain binding protein (14) . Using an affinity-purified polyclonal antiserum raised against human FACC, we have recently demonstrated that the FACC polypeptide is localized primarily to the cytoplasm (15) and suggested that it is unlikely to play a direct role in DNA repair. Utilizing another polyclonal antiserum, Yamashita et al. (16) have also localized the FACC polypeptide to the cytoplasm. In addition, these investigators have also detected 50- and 150-kDa polypeptides in lysates of radiolabeled, transformed lymphoblasts that immunoprecipitated with their antiserum. However, in view of possible fortuitous interactions of their antiserum with epitopes on unrelated proteins, the precise relationship of these proteins to FACC would be difficult to establish.

The genes for the remaining non-C complementation groups remain to be identified. Although the FA phenotype may result from distinct pathogenetic mechanisms, the overall similarity in phenotype suggests a biochemical mechanism characterized by a shared molecular defect, such as lesions in a multimeric complex or in a multistep metabolic pathway whereby the failure of any single component could lead to a similar dysfunction. The latter hypothesis predicts the occurrence of specific protein contacts involving FACC, particularly in the case of multimer formation. In an attempt to study this possibility, we have investigated the oligomeric structure of FACC by using a novel system for protein-protein interactions in vitro. Our results provide direct evidence for the binding of FACC to a family of cytoplasmic proteins. We also describe a rapid expression and selection technique for complementation of FA cells. Taken together, our results suggest that FACC forms an ubiquitous multimeric complex with at least three cytosolic polypeptides.


MATERIALS AND METHODS

Cell Culture

Cell lines were maintained as follows: COS-7 (green monkey kidney) in Dulbecco's modified essential medium (DMEM) (Life Technologies, Inc.) and 10% calf serum; Dami (human megakaryocytic leukemia), K562 (human chronic myeloid leukemia), and THP-1 (human monocytic leukemia) in RPMI 1640 medium (Life Technologies, Inc.) and 10% fetal calf serum; and human lymphoblastoid cells (RA 142, wild type for FACC; RA 568, FA complementation group C, compound heterozygous for a frameshift mutation resulting from a deletion of base 322 in exon 1, and an Arg Stop mutation in exon 6; RA 171 and GM4510, FA complementation group C, homozygous for a splice site mutation in intron 4 of the FACC gene; and RA 333 and RA 398, FA non-group-C cell lines) in RPMI 1640 medium and 15% fetal calf serum. All sera were heat-inactivated before use.

Generation of Antibody

Generation of a polyclonal antiserum against a recombinant fusion protein encoding glutathione S-transferase (GST) at the amino terminus and FACC (amino acids 6-558) at the carboxyl terminus has been described previously (15) . The immunoglobulin fraction from the rabbit antiserum was isolated by chromatography over a protein A-agarose column, which was depleted of antibodies directed against GST epitopes by extensive incubation with GST-bound glutathione-Sepharose 4B beads (Pharmacia Biotech Inc.). Affinity-purified anti-FACC antibody was obtained by chromatography of the GST-depleted fraction over an epoxy-activated silica column (Waters, Milford, MA) containing immobilized GST-FACC, followed by acid elution.

Construction of Epitope-tagged FACC

Based on the published nucleotide sequence of human FACC cDNA (8) , a cDNA clone from HL60 cells was isolated by reverse transcription and polymerase chain reaction (PCR) amplification (15) . This fragment was radiolabeled and used to screen a plasmid library from Raji cells in the episomal expression vector DRA-CD (17) by colony hybridization, and a 4.5-kb full-length clone that contains 1674 base pairs of coding sequence was obtained (called DRA-FACC). To facilitate subcloning, a 5` PCR primer was designed to include a site for SalI, and the 3` primer was modified at the termination codon of FACC to create a restriction site for BamHI. To minimize the possibility of mutation, we used Vent Polymerase (New England Biolabs, Beverly, MA) and 20 cycles of PCR amplification. The resulting 1.7-kb PCR fragment was digested with SalI and BamHI, mixed with an equal aliquot of a 0.7-kb BamHI- KpnI fragment encoding the constant region of the human IgG1 heavy chain (gift of B. Seed), and ligated into DRA linearized by SalI and KpnI. The resulting vector (called DRA-FACC1) contained the full-length coding sequence of human FACC at the amino terminus, a three-amino acid linker, and the cDNA for the heavy chain of human IgG1 at the carboxyl terminus containing the hinge and CHand CHdomains (see Fig. 1 ). Similarly, a 0.7-kb fragment encoding the mouse erythropoietin (EPO) cDNA was joined in-frame to the 0.7-kb IgG1 epitope and cloned into the expression vector pcDNA1 (Invitrogen, San Diego, CA) to generate EPO1.


Figure 1: Structure and expression of FACC constructs. A, the plasmid DRA-CD contains a hygromycin-resistance gene ( hph-76), the Epstein-Barr virus origin of replication ( OriP), Epstein-Barr virus nuclear antigen gene 1 ( EBNA-1) under the control of the SV40 late promoter, ampicillin resistance gene ( Amp), and the human CD4 cDNA under control of the HLA-DRA promoter (17). FACC or FACC1 inserts are expressed from the SV40 early promoter. B, to generate FACC1, the translated portion of FACC cDNA ( open bar) containing an artificial BamHI ( B) site at the 3` end in place of the natural termination codon was ligated in-frame to the constant region of the heavy chain cDNA of human IgG1 ( IgG1-Fc, solid bar). The sequence of the predicted fusion joint and the relative position of amino acid residues in the chimera are shown. aa, amino acids. C, oligomeric organization of FACC. FACC or FACC1 cloned in the mammalian expression vector pcDNA1 were expressed in COS-7 cells. After 48 h, metabolically labeled cytosolic extracts were incubated with an affinity-purified polyclonal anti-FACC antibody followed by immunoprecipitation with protein A-agarose beads. The bound protein was boiled in Laemmli buffer in the presence ( Reducing) or absence ( Non-reducing) of 100 mM dithiothreitol and analyzed on 10% SDS-polyacrylamide gels and with fluorography.



Transfection and Selection of FA Lymphoblasts

DRA-CD, DRA-FACC, or DRA-FACC1 was used to transfect GM4510 lymphoblastoid cells, which are homozygous for a splicing mutation in intron 4 of the FACC gene (9) . Briefly, 2.5 10cells were incubated with 20 µg of supercoiled DNA and 400 µg of Escherichia coli tRNA, and the mixture was subjected to electroporation using a Gene Pulser (Bio-Rad) at 260 V and 960 microfarads, as described previously (17) . For a rapid assessment of cell viability in response to MMC, transfected cells were selected by virtue of surface expression for the human T-cell antigen CD4. Parental B-lymphoblastoid cells do not express CD4 (data not shown). The anti-CD4 IOT4a monoclonal antibody (AMAC, Westbrook, ME) was bound to magnetic beads coupled to goat anti-mouse IgG (Advanced Magnetics, Cambridge, MA). Cells were incubated with 10 µl of slurry of washed beads and isolated by passage over a magnet. After incubation in complete medium overnight, MMC cytotoxicity assays were performed. In some experiments, cells sorted by Immunobead selection were further selected in hygromycin B (Boehringer Mannheim) at an initial dose of 125 µg/ml and up to 250 µg/ml, and after 2 weeks resistant cells were cloned by limiting dilution. All transfected cells were assessed by indirect immunofluorescence for FACC1 expression using fluorescein-conjugated goat anti-human IgG (Cappel, West Chester, PA).

COS Cell Transfection

COS-7 cells were cultured to 40-60% confluence in 10-cm dishes containing DMEM with 10% heat-inactivated calf serum. Plasmid DNA was resuspended to a final concentration of 1 µg/ml in 1.5 ml of DMEM with 10% newborn calf serum and containing 400 µg/ml DEAE-dextran and 100 µM chloroquine diphosphate. 16 h after transfection cells were detached with trypsin and replated in fresh DMEM with 10% calf serum. Most COS-7 cell transfections were performed with pcDNA1-based vectors.

Purification of Immunoglobulin Fusion Proteins Bound to Protein A-Agarose Beads

Monolayers of COS-7 cells transfected with immunoglobulin fusion constructs were processed after 48 h by washing with cold PBS followed by lysis in buffer A. Postnuclear supernatants were incubated with agarose beads coupled to protein A (Bio-Rad) for 16 h at 4 °C. Approximately 200 µl of a 50% slurry of beads were incubated with lysates from eight 10-cm dishes. Beads were washed three times with NET-gel buffer containing 100 mM dithiothreitol and three more times in NET-gel buffer containing 0.3 M NaCl. Beads stored in PBS containing 100 mM dithiothreitol were active for at least 3 months as assessed by the binding assay. The concentration of bound protein was estimated using the BCA assay (Pierce) after elution with 4 M imidazole. Eluted protein was equilibrated in PBS and concentrated using Centricon-30 spin columns (Amicon, Beverly, MA), and small aliquots were analyzed by electrophoresis on 10% SDS-polyacrylamide gels and Coomassie Blue staining.

Isolation of Cellular Fractions

For large scale preparation of subcellular fractions, 100 10Dami cell pellets was harvested and metabolically labeled with ExpreSS label (0.2 mCi/ml; DuPont NEN) for 2 h in cysteine- and methionine-deficient DMEM. Cell pellets were resuspended in a buffer containing 0.25 M sucrose and 10 mM Hepes (pH 7.2) and disrupted by 20 strokes in a Dounce homogenizer (type B) in the presence of a mixture of protease inhibitors, including 100 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml aprotinin. Nuclei were sedimented for 5 min at 1000 g, washed in ice-cold PBS, and disrupted as described previously (15) . The crude cytosolic slurry was subjected to further fractionation by layering onto a discontinuous 5-50% sucrose gradient and by centrifugation for 3 h at 80,000 g using a Beckman SW 41 rotor. The pellet was enriched with mitochondria. The supernatant was subjected to a final round of centrifugation for 1 h at 100,000 g to isolate total membranes (pellet) and cytosol (supernatant). Alternatively, for small scale experiments, metabolically labeled cells were washed with PBS and lysed in buffer A (20 mM Tris, pH 8.0, 50 mM NaCl, 0.5% deoxycholate, 1% Nonidet P-40) in the presence of protease inhibitors. Nuclear and crude cytosolic preparations were obtained by centrifugation of the lysates for 5 min at 1000 g, as described previously (15) .

Immunoprecipitation and Co-precipitation

For co-precipitation experiments with immunoglobulin fusion proteins bound to protein A-agarose beads, cellular fractions from either large scale or small scale preparations were adjusted to a final concentration of 0.2% Nonidet P-40 and 50 mM NaCl, whereas immunoprecipitations with anti-FACC antibody were performed in a buffer containing 150 mM NaCl and 1% Nonidet P-40. Extracts were incubated sequentially with anti-FACC antibody followed by protein A-agarose beads (Bio-Rad), with beads alone, or with beads that were previously bound to FACC1 or EPO1. Incubations with the primary antibody or recombinant protein were typically for 16 h at 4 °C, whereas incubation with secondary beads was limited to 2 h at 4 °C. Immunocomplexes were washed three times with NET-gel buffer over 15 min and boiled in 1 Laemmli buffer in the presence or absence of 200 mM dithiothreitol (18) . Samples were subjected to electrophoresis on denaturing 10% SDS-polyacrylamide gels, fixed in acetic acid, and analyzed by fluorography using 22% 2,5-diphenyloxazole (Sigma). For quantitation of the immunoprecipitated products, appropriate regions of the autoradiogram corresponding to FACC or the FABPs were excised, shredded into small pieces, dissolved in scintillation fluid (OptiPhase HiSafe 3, LKB Scintillation Products, Leicster, United Kingdom), and counted on a Wallac 1409 liquid scintillation counter (Pharmacia).

Immunoblot Analysis

Cells were washed in PBS, and equal numbers were lysed directly into Laemmli sample buffer containing 100 mM dithiothreitol (18) . After electrophoresis on SDS-polyacrylamide gels, the protein was transferred to polyvinyldifluoride membranes as described by the manufacturer and blocked in BLOTTO containing 5% nonfat milk, and individual strips were incubated either with purified FACC1 (50 µg/ml) or human IgG (50 µg/ml). After washing in Tris-buffered saline containing 0.1% Tween, bound immunocomplexes were reacted with horseradish peroxidase-conjugated goat anti-human IgG or anti-rabbit IgG, as appropriate, and visualized by chemiluminescence according to the directions of the manufacturer (DuPont NEN). In some experiments, purified GST or GST-FACC was included as a competitor, as described previously (15) .

Determination of Cell Survival

For measurements of cell survival in response to MMC (Sigma), 2 10exponentially growing cells were incubated in different concentrations of MMC (up to 250 ng/ml) for 7 days, and viable cells were counted by trypan blue exclusion.


RESULTS

Structure of FACC and FACC1

Screening of 50,000 clones of the Raji cDNA library constructed in the vector DRA-CD (Fig. 1 A) by colony hybridization yielded two clones that hybridized to radiolabeled human FACC cDNA. One of these plasmids contained a 4.5-kb insert, which had a pattern of restriction endonuclease sites identical to a full-length FACC cDNA clone described previously (8) . The FACC1 insert (Fig. 1) was cloned in DRA-CD as well, and, for expression in COS-7 cells, both inserts were also cloned in pcDNA1. The presence of the hinge region in FACC1 predicts that the chimeric molecule would form dimers under non-reducing conditions.

Oligomeric Structure of FACC

Metabolic labeling of COS-7 cells transfected with FACC or FACC1 and immunoprecipitation with an affinity-purified polyclonal antiserum directed against FACC showed predominant bands at 63 and 85 kDa, respectively, under reducing conditions, consistent with the predicted size of these polypeptides (Fig. 1 B). Because FACC contains a number of cysteine residues, the possibility of dimer formation via disulfide bridging was evaluated by electrophoresis of the immunocomplexes under non-reducing conditions. There was no retardation in the mobility of FACC immunocomplexes, suggesting that FACC primarily forms monomers under non-reducing conditions. By contrast, as predicted, most of FACC1 formed dimers as judged by the slower migration of immunocomplexes.

Expression and Function of Chimeric FACC

We reasoned that the use of a functionally competent form of FACC for an in vitro binding assay may be more physiologically meaningful. We tested the ability of FACC1 to correct the cellular hypersensitivity of GM4510 cells to MMC. After electroporation of these cells with DRA-CD, DRA-FACC, or DRA-FACC1 and Immunobead selection, expression of FACC was assessed initially by indirect immunofluorescence. In both FACC- and FACC1-transfected cells, over 90% of the cells showed cytoplasmic staining (data not shown), similar to our results described previously (15) . Transfected cells were divided into two aliquots; a small portion was tested for MMC hypersensitivity without further selection, and the remainder was selected for resistance to hygromycin B. Expression of FACC1 was analyzed in the latter population by metabolic labeling and immunoprecipitation with protein A-agarose. A polypeptide of 85 kDa was detected in both transiently transfected COS-7 and stably transfected GM4510 cells with FACC1-containing plasmids but not in cells transfected with control vectors alone (Fig. 2 A). GM4510 cells stably expressing FACC and FACC1 were significantly more resistant to MMC-induced cytotoxicity than hygromycin-resistant control cells transfected with the DRA-CD plasmid alone (Fig. 2 B). The doses of MMC reducing survival to 50% of control levels (EC) were 7.5 (DRA-CD-transfected cells), 90.5 (DRA-FACC-transfected cells), and 102.5 ng/ml (DRA-FACC1-transfected cells). Transfected GM4510 cells selected either by immunomagnetic sorting alone or with additional selection in hygromycin B showed no significant differences in the MMC sensitivity. Thus, FACC1 expression was able to correct the hypersensitivity of these cells to approximately the same degree as FACC, as described previously (8) . Furthermore, the use of DRA-CD vectors containing FACC constructs and immunomagnetic selection for surface CD4 expression provided a rapid test of complementation for this group of FA.


Figure 2: Expression and function of chimeric FACC. A, COS-7 cells transfected with pcDNA1 ( lane 1) or pcDNA1-FACC1 ( lane 2) and GM4510 cells transfected with DRA-CD ( lane 3) or DRA-FACC1 ( lane 4) were metabolically labeled and analyzed by single-step immunoprecipitation with protein A-agarose beads. The bound protein was eluted under reducing conditions and analyzed by SDS-polyacrylamide gel electrophoresis. B, complementation of FA-C cells. GM4510 lymphoblastoid cells transfected with DRA-CD ( GM-CD, ), DRA-FACC ( GM-FA, ) or DRA-FACC1 ( GM-FAIg, ) by electroporation were selected after 72 h by immunomagnetic sorting for surface CD4 expression. Cellular viability was assessed by trypan blue exclusion (mean of three values) after continuous growth in MMC at the indicated doses for 7 days. These results were not significantly different from the sensitivity of cells selected further for resistance to hygromycin B (data not shown).



Identification of Cytosolic Proteins That Bind to FACC

FACC1 or EPO1 was overexpressed in COS-7 cells. After 48-72 h, the chimeric proteins in cellular lysates were purified to near homogeneity by immobilization onto protein A-agarose beads and extensive washing. The bound polypeptides were then used as baits for the detection of proteins that bind to FACC but not to EPO or to the IgG1 epitope. RA142 human lymphoblastoid cells (non-FA) were metabolically labeled, and cell lysates were fractionated into nuclear and cytosolic components. Extracts were then incubated with various protein A-agarose beads, and bound radiolabeled proteins were analyzed by SDS-polyacrylamide gel electrophoresis. In these experiments the matrix retains a limited number of common background bands. However, three polypeptides of 65, 50, and 35 kDa (called FABP-65, FABP-50, and FABP-35, respectively) were detected uniquely with FACC1-bound beads in cytosolic extracts (Fig. 3). FABPs failed to bind under conditions of higher ionic strength (100-250 mM NaCl) or detergent concentrations (1% Nonidet P-40; data not shown). Similarly, no FABPs were detected with the use of radiolabeled nuclear extracts (Fig. 3). In order to exclude potential interactions via the IgG1 domain, lysates were also incubated with EPO1, an unrelated chimeric IgG1 molecule bound to protein A-agarose. Unlike FACC1, no binding to FABPs was observed with the use of EPO1. These polypeptides were also detected in two different lymphoblastoid cell lines from FA patients with documented FACC mutations, as well as in two additional FA cell lines that belong to non-C complementation groups (Fig. 3).


Figure 3: Identification of cytosolic FACC-binding proteins. Analysis of S-labeled lysates from non-FA and FA lymphoblastoid cells incubated with either FACC1 or EPO1 bound to protein A-agarose is shown. MMC (1-100 ng/ml; results shown are for 1 ng/ml) was added to cells in some experiments prior to metabolic labeling or during in vitro immunocomplex formation, as shown. Cytosolic and nuclear extracts were prepared as described previously (15). The genotype of the cells is shown at the top of the figure. The positions of FACC-binding proteins are shown ( arrows).



The co-precipitation assay was also used to survey a number of mammalian cell lines for expression of FABPs. After metabolic labeling, crude cytosolic extracts were incubated with FACC1-bound beads and analyzed by SDS-polyacrylamide gel electrophoresis. Despite differences in the background bands, all three FABPs were detected in every cell line examined that was wild type for FACC (Fig. 4). These data demonstrate that FACC binds to at least three cytosolic, but not nuclear, proteins in vitro, and, similar to FACC, the FABPs are also most likely expressed ubiquitously. The interaction of these polypeptides with FACC suggests the formation of a multimeric complex in vitro.


Figure 4: Survey of S-labeled lysates from Dami ( lanes 1 and 2), THP-1 ( lanes 3 and 4), K562 ( lanes 5 and 6), and COS-7 cells ( lanes 7 and 8) analyzed by SDS-polyacrylamide gel electrophoresis. Lysates were incubated with either unbound ( lanes 1, 3, 5, and 7) or FACC1-bound ( lanes 2, 4, 6, and 8) protein A-agarose. The positions of the FACC-binding proteins are shown ( arrows).



Co-fractionation of FACC and the FABPs

In order to demonstrate directly that FACC co-fractionates with the FABPs, subcellular fractions were obtained from 100 10metabolically labeled Dami cells and assayed, in parallel experiments, by immunoprecipitation with either anti-FACC antibody or FACC1-bound protein A-agarose beads. Both FACC and the FABPs co-fractionated exclusively with cytosolic and, to a lesser extent, membrane extracts ().

Detection of FACC-binding Polypeptides by Immunoblotting

In order to increase the validity of detection of the FABPs by the co-precipitation assay, we also attempted to detect these polypeptides by an independent assay. We obtained 200 µg of purified FACC1 polypeptide from sixty 10-cm dishes of COS-7 cells (3 10cells) transiently transfected with pcDNA-FACC1 (Fig. 5 A). As visualized by Coomassie Blue staining, isolation by protein A-agarose chromatography also resulted in the co-purification of IgG; this was also noted in lysates from pcDNA1-transfected cells (data not shown). Immunoblots of Dami cell lysates were incubated with purified FACC1 or total human IgG, followed by incubation with horseradish-conjugated goat anti-human IgG and detection by chemiluminescence (DuPont NEN). Specific bands at 65, 50, and 30 kDa (Fig. 5 B) matched the sizes of FABPs detected by the co-precipitation assay. By contrast, immunoblot strips incubated with anti-FACC antibody showed a single band at 63 kDa, consistent with the predicted size of FACC, which is competed with the addition of GST-FACC fusion protein but not GST protein (15) . These observations strongly suggest that the FABPs are antigenically distinct from FACC.


Figure 5: Detection of FACC-binding proteins by ligand blotting. A, equal numbers of parental COS-7 cells ( lane 1) and cells transfected with either pcDNA1 ( lane 2) or pcDNA-FACC1 ( lane 3) and a 10-µg aliquot of purified FACC1 ( lane 4) were subjected to electrophoresis on a 10% SDS-polyacrylamide gel and visualized by Coomassie Blue staining. The positions of FACC1 as well as the heavy ( Ig-H) and light ( Ig-L) chains of IgG are shown. B, immunoblot analysis of Dami cell lysates probed with FACC1 (50 µg/ml) followed by incubation with goat anti-human IgG coupled to horseradish peroxidase. After washing, blots were developed by a chemiluminescence method (Reflection, DuPont NEN). Purified GST-FACC (200 µg/ml, lane 2) or GST (200 µg/ml, lane 3) was added as a competitor. The positions of FABPs are shown.




DISCUSSION

Observations of elevated frequencies of spontaneous chromosomal aberrations and induction of such lesions with DNA bifunctional alkylating agents (1, 5, 19) have generally led to the classification of FA with other inherited disorders characterized by chromosomal instability or abnormal DNA repair. The recent cloning of the FACC gene by virtue of its ability to correct the cross-link-induced hypersensitivity of FA cells (8) and identification of mutant alleles in the DNA of FA-C patients (8, 9, 10, 11, 12) have confirmed the central role of the FACC polypeptide in the pathobiology of FA and provided an opportunity to understand the fundamental cellular and biochemical defects that underlie this disorder. The ubiquitous detection of FACC transcripts in most tissues and cell lines (Ref. 8 and data not shown) has raised the possibility of a ``housekeeping'' function for the protein. However, a putative role in DNA repair has been difficult to establish, particularly in view of recent observations that the FACC polypeptide is primarily cytoplasmic (15, 16) . Further understanding of the subcellular milieu and identification of proteins that interact with FACC could help to elucidate its biochemical function. Such an approach could also lead to an understanding of the possible relationship of the gene products from different complementation groups to each other.

In this study, we have developed a direct in vitro assay for the detection of proteins that interact specifically with FACC. A number of assays for protein-protein interactions have been described previously, including biochemical methods, such as the use of bacterial fusion proteins immobilized to solid matrices (see Ref. 20 for an example) as well as genetic assays (see Ref. 21 for an example). A common theme is the design of an epitope, or ``bait,'' that is used to capture binding proteins. We adapted an expression system using mammalian cells for several reasons. First, little is known about the nature of post-translational modifications, if any, that might regulate the function of FACC. For example, there are a number of consensus motifs in FACC for Ser/Thr phosphorylation, although their functional role is not yet clear. A mammalian expression system is more likely to yield a ligand that contains the correct post-translational modifications. Second, it would be desirable to design a ligand that recapitulates the biological function of interest. However, as there are no in vitro assays available at present for the function of the FACC polypeptide, our strategy of establishing the complementation activity of FACC1 in FA-C cells may be a reasonable compromise in the design of such a reagent. Finally, the immunoglobulin tag greatly simplifies the isolation of the polypeptide from cell lysates without resorting to complex purification schemes, which could lead to inactivation or degradation of the protein of interest. Indeed, the carboxyl-terminal location of the immunoglobulin epitope and binding of the chimeric protein to protein A are somewhat helpful features in evaluating the overall integrity of the purified polypeptide.

The use of both co-precipitation and immunoblotting assays demonstrates the same panel of polypeptides that bind to FACC, at least as judged by their sizes. FABP-50 appears to be more abundant by both the co-precipitation and ligand blotting assays. Alternatively, this could result from differences in the stoichiometry of binding of FABP-50, versus other FABPs, to FACC. A previous study (16) reported an association of FACC with 50- and 150-kDa polypeptides (called FACC-related proteins) using an independently derived polyclonal antiserum against human FACC. This antiserum also detects a number of additional bands, and no immunoblotting studies were reported to evaluate the possibility of shared antigenicity between these proteins. Thus, the relationship of FACC to FACC-associated proteins in this system is not altogether clear. In our experience, the affinity-purified anti-FACC antibody described here detects a single band corresponding to the predicted size of FACC, which is competed by the addition of purified GST-FACC protein (15) . Furthermore, our experiments differ from those reported by Yamashita et al. (16) in two fundamental ways. First, we have utilized the recombinant FACC protein, not the anti-FACC antiserum, to identify heterologous proteins that bind to it directly. Thus, the possibility that an antiserum capable of immunoprecipitating heterologous proteins fortuitously is obviated. Second, the combined immunoprecipitation and immunoblotting studies clearly demonstrate that the FABPs are antigenically distinct from FACC. Precautions were taken against proteolysis (see ``Materials and Methods''), and the absence of smaller species (specifically 50- and 35-kDa polypeptides) in immunoprecipitates of FACC performed under conditions similar to the co-precipitation assays argues against the spurious proteolysis of FACC in our assays. However, the possibility that FABP-50 or FABP-35 is derived from FABP-65 cannot be excluded completely. We do not believe that this is the case, because metabolic labeling of either lymphoblasts or Dami cells for 15 min instead of 1 h followed by cold chase for 2 h appeared to make little difference in the co-precipitation pattern (data not shown). Thus, a post-translational mechanism for generating the smaller FABPs from a common precursor seems unlikely.

Taken together, these results suggest that FACC forms a multimeric complex in vitro. The absence of higher molecular weight oligomers in non-reduced immunocomplexes and the requirement for low ionic strength in the co-precipitation assays suggest that these interactions are mediated by non-covalent forces rather than mechanisms that involve disulfide bridging or other covalent interactions.

Although FACC is primarily localized to the cytoplasm (15, 16) , previous studies have implicated both nuclear and non-nuclear proteins in the pathogenesis of FA, including poly(ADP)-ribosyltransferase (22, 23) , DNA topoisomerase I (24, 25) , a putative endonuclease (26) , and a mitochondrial nuclease (27) . Thus, it was of interest to determine the subcellular localization of FABPs. All three FABPs were detected with the use of cytosolic and membrane, but not nuclear extracts, and showed no detectable changes after treatment of cells with mitomycin C. These results suggest that macromolecular complexes exist constitutively as preformed structures. The similarity in phenotype of the different FA complementation groups (1) could result from a number of different possibilities, including direct interactions or interdependence of their respective polypeptides for activity. The formation of multimeric complexes is one way of accounting for this possibility. Specific interactions between DNA repair proteins have been noted previously (28, 29) . By analogy, it is possible the gene products that are deficient in FA complementation groups also can interact with each other. Although we were able to detect FABPs in cell lysates from non-C groups of FA, the presence of subtle mutations in the FABP genes or an alteration in the function of the putative multimeric complex cannot be excluded.

Two very recent observations lead us to suggest a possible functional role for this macromolecular complex. First, the ability of FACC to correct the MMC hypersensitivity of a FA-C lymphoblast cell line is compromised by its forced localization to the nucleus.() Thus, the cytoplasmic location of FACC appears to be critical for its ability to ameliorate the cytotoxic effects of bifunctional cross-linkers. Second, direct DNA cross-linking experiments suggest that the function of FACC precedes the formation of interstrand DNA cross-links.() Therefore, in this experimental system FACC can act as a cellular guard against genotoxic agents. These observations lead us to suggest that one function for the FABPs may be as cytoplasmic anchors for FACC, a hypothesis that can be tested directly after the isolation of the FABP genes. The ligand blotting assay (Fig. 5 B) provides the experimental framework for the functional isolation of FABP genes by binding of recombinant clones in cDNA expression libraries to FACC1; this work is currently in progress.

Finally, the combined use of mammalian expression vectors that undergo episomal replication and selection for a specific surface antigen encoded by the vector should greatly facilitate the complementation analysis of FA cells and possible ascertainment for group C. In our studies, the time elapsed from the beginning of electroporation to the completion of MMC cytotoxicity assays was approximately 11 days. This strategy would be particularly useful in the evaluation of potential regulatory mutations in FACC alleles where the coding region has been demonstrated to be free of mutations or in cases of suspected polymorphisms (9, 12) .

  
Table: Subcellular distributions of FABP-65, FABP-50, FABP-35, and FACC in Dami cells

The fractions were isolated as described under ``Materials and Methods.'' The values are the means ± S.D. of four experiments.



FOOTNOTES

*
This work was supported by a grant from the Henry M. and Lillian Stratton Foundation (to H. Y.), by Grant 6-FY95-0279 from the March of Dimes Birth Defects Foundation (to H. Y.), and by Grant HL32987 from the National Institutes of Health (to A. D. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Brigham and Women's Hospital, 221 Longwood Ave., LMRC 620, Boston, MA 02115. Tel.: 617-732-5464; Fax: 617-739-3324.

The abbreviations used are: FA, Fanconi anemia; FA-C, Fanconi anemia group C; PCR, polymerase chain reaction; FACC, Fanconi anemia complementation group C; FABP, FACC-binding protein; GST, glutathione S-transferase; MMC, mitomycin C; EPO, erythropoietin; DMEM, Dulbecco's modified essential medium; kb, kilobase pair(s); PBS, phosphate-buffered saline.

H. Youssoufian, unpublished observations.

H. Youssoufian and H. Joenje, unpublished observations.


ACKNOWLEDGEMENTS

-We thank the laboratory members of Drs. H. Franklin Bunn and Robert I. Handin for stimulating discussions throughout the course of this study.


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