From the
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 (FACC
Fanconi anemia (FA)
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
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.
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 FACC
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.
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) .
The fractions were
isolated as described under ``Materials and Methods.'' The
values are the means ± S.D. of four experiments.
-We thank the laboratory members of Drs. H.
Franklin Bunn and Robert I. Handin for stimulating discussions
throughout the course of this study.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1). Expression of FACC
1 in FACC-deficient Fanconi
anemia (FA) lymphoblasts corrected the hypersensitivity of these cells
to mitomycin C. Binding of FACC
1 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 FACC
1 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 FACC
1 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.
(
)
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.
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.
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 CH
and CH
domains (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 EPO
1.
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 FACC
1, 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 FACC
1 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
10
cells 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 FACC
1 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 10
Dami
cell pellets was harvested and metabolically labeled with
Expre
S
S 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 EPO
1. 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 10
exponentially 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.
Structure of FACC and FACC
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
1 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
FACC
1 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
FACC
1 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-FACC
1 and Immunobead
selection, expression of FACC was assessed initially by indirect
immunofluorescence. In both FACC- and FACC
1-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 FACC
1 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 FACC
1-containing plasmids but not in
cells transfected with control vectors alone (Fig. 2 A).
GM4510 cells stably expressing FACC and FACC
1 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-FACC
1-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, FACC
1 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-FACC
1 ( 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-FACC
1
( 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 EPO
1 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 FACC
1-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 EPO
1, an unrelated
chimeric IgG1 molecule bound to protein A-agarose. Unlike FACC
1,
no binding to FABPs was observed with the use of EPO
1. 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 FACC
1 or EPO
1
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
FACC
1-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 10
metabolically labeled Dami cells and assayed, in parallel
experiments, by immunoprecipitation with either anti-FACC antibody or
FACC
1-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 FACC
1 polypeptide from sixty 10-cm dishes of COS-7
cells (
3
10
cells) transiently transfected
with pcDNA-FACC
1 (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 FACC
1 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 FACC
1 ( lane 4) were subjected to
electrophoresis on a 10% SDS-polyacrylamide gel and visualized by
Coomassie Blue staining. The positions of FACC
1 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
FACC
1 (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.
1 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.
(
)
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 FACC
1; this
work is currently in progress.
Table: Subcellular distributions of FABP-65,
FABP-50, FABP-35, and FACC in Dami cells
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.