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Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2229-2236
The Fanconi Anemia Proteins FAA and FAC Function in Different Cellular
Compartments to Protect Against Cross-Linking Agent Cytotoxicity
By
Frank A.E. Kruyt and
Hagop Youssoufian
From the Department of Molecular and Human Genetics and the
Department of Medicine, Baylor College of Medicine, One Baylor Plaza,
Houston, TX.
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ABSTRACT |
Fanconi anemia (FA) is an autosomal recessive disease characterized
by chromosomal instability, bone marrow failure, and a high risk of
developing malignancies. Although the disorder is genetically
heterogeneous, all FA cells are defined by their sensitivity to the
apoptosis-inducing effect of cross-linking agents, such as mitomycin C
(MMC). The cloned FA disease genes, FAC and FAA, encode
proteins with no homology to each other or to any known protein. We
generated a highly specific antibody against FAA and found the protein
in both the cytoplasm and nucleus of mammalian cells. By subcellular
fractionation, FAA is also associated with intracellular membranes. To
identify the subcellular compartment that is relevant for FAA activity,
we appended nuclear export and nuclear localization signals to the
carboxy terminus of FAA and enriched its localization in
either the cytoplasm or the nucleus. Nuclear localization of FAA was
both necessary and sufficient to correct MMC sensitivity in FA-A cells.
In addition, we found no evidence for an interaction between FAA and
FAC either in vivo or in vitro. Together with a previous finding that
FAC is active in the cytoplasm but not in the nucleus, our results
indicate that FAA and FAC function in separate subcellular
compartments. Thus, FAA and FAC, if functionally linked, are more
likely to be in a linear pathway rather than form a macromolecular
complex to protect against cross-linker cytotoxicity.
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INTRODUCTION |
FANCONI ANEMIA (FA) is an autosomal
recessive disease that frequently leads to bone marrow failure and
myeloid leukemias at childhood.1-3 Cells from FA patients
are characterized by hypersensitivity to cross-linking agents, such as
mitomycin C (MMC) and diepoxybutane, and a propensity to
apoptosis.4-7 By somatic cell fusion studies, eight
different complementation groups of FA have been identified, named A to
H,8-10 suggesting that the inactivation of at least eight
distinct genes could give rise to the clinical and cellular phenotype
recognized as FA. One of the central questions in FA research is to
elucidate the relationship, if any, of these genes and their products
to each other. To date two FA genes, FAC and FAA, have
been cloned.11-13 The proteins encoded by these genes are
unique and show no homology to each other.
There is conflicting evidence about the possible involvement of FA
proteins in DNA repair.3 The FAC protein has been shown to
localize predominantly to the cytoplasmic compartment14,15 and function in a prerepair pathway.16 In this capacity,
cytoplasmic localization of FAC is essential for cross-linker
complementation of FA group C (FA-C) cells, while forced nuclear
localization completely abolishes this activity.16 In FAA,
the presence of a putative nuclear localization signal (NLS) in the
amino-terminal region suggests a nuclear function for this
protein.12,13 However, in preliminary studies, a chimeric
molecule consisting of full-length FAA tagged with the green
fluorescent protein (GFP) showed a predominantly cytoplasmic
localization in human 293 cells, and inactivation of the NLS motif by
site-directed mutagenesis did not abort the complementation function of
FAA.17 Although this result suggested that FAA also has a
cytoplasmic role, the inclusion of GFP and potential effects on the
localization of the protein precluded any definitive conclusions about
the localization and function of wild-type FAA.
The participation of two or more FA proteins in a single macromolecular
complex that regulates genomic stability is a plausible mechanism that
could account for the phenotypic similarity of different FA
complementation groups. Previous studies using extracts derived from
human lymphoblasts and the human megakaryocytic cell line Dami showed
that FAC binds to at least three ubiquitous cytoplasmic proteins.18 However, the expression and size of these
FAC-binding proteins in lymphoblasts from FA complementation group A
(FA-A) were normal, and there was no interacting protein that, in
retrospect, matched the molecular size of FAA. Another possibility is
that FA proteins are linked in a linear pathway, and constituents of the pathway may be localized in distinct cellular compartments. The
identification of a second FA protein now enables us to test these
possibilities.
In this study, we report the generation of an antibody against FAA that
is able to detect FAA by various biochemical assays. By
immunofluorescence studies, FAA localizes to both the cytoplasmic and
nuclear compartment of human and other mammalian cells and shows a
clearly different intracellular distribution compared with FAC. We
found no evidence for an interaction between the two FA proteins both
in vitro and in vivo by coprecipitation assays that have been
successfully applied to demonstrate other protein interactions with
FAC.18,19 Finally, we provide strong evidence that FAA must
reside in the nucleus to protect against cross-linker cytotoxicity. Our
data support a model in which FAC and FAA function in different
cellular compartments to defend the genome against cross-linking
agents.
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MATERIALS AND METHODS |
Cell culture, transfection, and MMC sensitivity assay.
Lymphoblasts were maintained in RPMI 1640 medium containing 10% fetal
calf serum (FCS). Stably transfected lymphoblasts were grown in the
same medium supplemented with 200 µg/mL hygromycin. HeLa, COS-1, and
293 cells were grown in Dulbecco's Minimal Essential Medium (DMEM;
GIBCO-BRL, Grand Island, NY) with 10% FCS. COS-1 cells were
transfected using DEAE-dextran as described previously18 and HeLa and 293 cells were transfected using Superfect (QIAGEN, Valencia, CA) according to the manufacturers' instructions. Stably transfected lymphoblasts were generated by electroporation as described
previously.16 The MMC growth inhibition assay was performed
by exposing parallel cultures (7.5 × 104 cells per
mL) to various concentrations of MMC. Cell numbers were determined
using a Coulter counter after the untreated control cells had undergone
three or more cell divisions.
Constructs.
For the generation of an affinity-purified antibody against FAA, His-
and GST-tagged FAA were made as follows: The amino terminal portion of
FAA encompassing amino acids 2 to 321 was amplified from pREP4-FAA
using the primers FA105 (5 -CGGGATCCGACTCGTGGGTCC-3) and
FA106 (5-CGGAATTCGTC GACTGAAGAACCTCTTCA-3). The resulting fragment of approximately 1 kb was digested with BamHI and
Sal I and subcloned into the corresponding sites of pQE30
(QIAGEN, Santa Clarita, CA) to generate an open reading
frame (ORF) for an approximately 37-kD His-FAA fusion
protein. The same polymerase chain reaction (PCR) fragment cut with
BamHI and EcoRI and subcloned into pGEX2TK yielded
pGST-FAA1 encoding an approximately 62-kD fusion protein. Sequences
3 of the stop codon of FAA were deleted by PCR using the primers
FA-3X
(5-TCAGTCTAGATTATTCAGAAGAGATGAGGCTCCTGGGACAGGT-3) and FA-101 (5-CTTAATTTTGACCTCTGCTCTGGTGTG-3), and the
resulting fragment was subcloned into pcDNA3 (Invitrogen, San Diego,
CA) or pDR2 (CLONTECH, Palo Alto, CA) to generate pcDNAFAA and pDRFAA, respectively. A wild-type or a mutant nuclear export signal (NES) was
appended to FAA in several stages. A 2.9-kb Kpn
I-HindIII fragment of FAA derived from the earlier described
FAA-GFP construct17 was subcloned in pBluescript KS. After
digestion with HindIII the annealed oligonucleotides NES1
(5-AGCTTAATGAATTAGCCTTGAAATTAGCAGGTCTTGATATCAACGT-3) and NES2
(5-CTGACGTTGATATCAAGACCTGCTAATTTCAAGGCTAATTCATTA-3) were inserted. After additional subcloning steps, the FAA ORF was
reconstructed in pcDNA3 and pDR2 to yield pcDNAFAA-NES and pDRFAA-NES,
respectively, which encode FAA fused to wild-type NES. In a similar
way, pcDNA- and pDRFAA-P12 were constructed by using primers similar to
the NES primers described above in which the underlined nucleotides in
NES1 and NES2 were substituted for GC to generate FAA fused to mutant
NES. Nuclear-targeted FAA expression plasmids were made following a
similar strategy as described above for NES-fusion constructs using the
oligonucleotides NLS1 (5-AGCTTCCGAAGAAAAAGAGAAAGGTGGT-3),
NLS2 (5-CTAGACC ACCTTTCTCTTTTTCTTCGGA-3), mutNLS1
(5-AGCTTCCGAAAGACAAAGAG AAAGGTGGT-3), and mutNLS2
(5-CTAGACCACCTTTCTCTTTGTCTTCGGA-3). The pED6FAC 1
construct has been described.18
Generation of affinity-purified FAA antibody.
Rabbits were immunized against the His-FAA1 protein that was purified
from Escherichia coli strain M15 using nickel (Ni)-agarose beads (QIAGEN) according to standard procedures. Affinity-purification of the FAA antibody was performed by passage over a column containing GST-FAA1 bound to glutathionine-Sepharose 4B beads (Pharmacia Biotech,
Piscataway, NJ) as described.18,20
Immunofluorescence microscopy and subcellular fractionation.
For colocalization experiments, HeLa and 293 cells were transiently
transfected with pcDNAFAA and pED6FAC1, and replated on glass
coverslips after 24 hours. After another 24 hours, cells were processed
for immunofluorescence microscopy as described before.15
Conjugated antibodies used were fluorescein-conjugated goat-anti-rabbit and rhodamine-conjugated goat-anti-human IgG (Cappel
Laboratories, West Chester, PA). To visualize nuclei, cells were
stained with bisbenzimide (Sigma Chemical Co, St Louis, MO).
Subcellular fractionation steps were performed at 4°C, and all
buffers were supplemented with the protease inhibitor
phenylmethylsulphonyl-fluoride (PMSF; final concentration 0.5 mM).
Transiently transfected or nontransfected cells cultured in 150-mm
dishes were rinsed with phosphate-buffered saline and lysed in 1 mL
lysis buffer, containing 20 mmol/L Tris-HCl, pH 8.0, 50 mmol/L NaCl, 1 mmol/L EDTA, and 0.5% Nonidet P-40 (NP-40). After 15 minutes, nuclei
were pelleted by centrifugation for 5 minutes at 600g and the
supernatant was taken as the crude cytoplasmic fraction. After
centrifugation of this fraction for 45 minutes at 100,000g, the
supernatant (cytosolic fraction) was removed and the pellet consisting
of intracellular membranes was resuspended in 250 µL lysis buffer,
briefly sonicated, and centrifuged to remove insoluble debris. Nuclei
were washed twice in lysis buffer and resuspended in 500 µL lysis
buffer supplemented with NaCl to a final concentration of 250 mmol/L,
disrupted by shearing with a syringe using a 21-gauge needle, and
briefly sonicated. Debris was pelleted and the supernatant was
designated as the nuclear extract.
Western blotting, metabolic labeling, and immunoprecipitation.
Western blotting, 35S-methionine labeling of cells, and
immunoprecipitation were performed as described
earlier.15,18 Cells were lysed in low salt buffer (20 mmol/L Tris-HCl, pH 8.0, 50 mmol/L NaCl, 2 mmol/L EDTA, 0.1% NP-40),
in medium salt buffer (20 mmol/L Tris-HCl, pH 8, 100 mmol/L NaCl, 2 mmol/L EDTA, 0.5% NP-40), or in RIPA buffer (50 mmol/L Tris-HCl, pH
7.5, 150 mmol/L NaCl, 1 % NP-40, 0.5% sodium deoxycholate, 0.1%
sodium dodecyl sulfate [SDS]), which were supplemented with the
protease inhibitor PMSF and the phosphatase inhibitor sodium
orthovanadate at final concentrations of 0.5 mmol/L and 1 mmol/L,
respectively. For Western blotting, lysates in low salt buffer
representing the equivalent of 2 × 105 cells per lane
were subjected to electrophoresis on an 8% polyacrylamide gel
(SDS-PAGE) and transferred to Polyscreen membrane (NEN Life Science
Products, Boston, MA). Blots were incubated with affinity-purified FAA
antibody, anti- -tubulin antibody (Boehringer Mannheim Biochemicals, Indianapolis, IN) or anti-human topoisomerase II antibody (GenoSys, Houston, TX) in TBST (10 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 0.05% Tween 20) supplemented with 4% nonfat dry milk and 1% bovine serum albumin, followed by incubation in the same buffer with either
peroxidase-conjugated goat-anti-rabbit IgG or goat-anti-mouse IgG
(GIBCO-BRL) and developed by using enhanced chemiluminescence (Amersham
Life Sciences, Arlington Heights, IL). For immunoprecipitation, 1.5 µg purified anti-FAA or anti-FAC antibody was added per sample of
cell lysates in either medium salt buffer or RIPA buffer. After incubation for 1 hour at 4°C with rotation, protein A slurry
(Bio-Rad Laboratories, Richmond, CA) or protein A/G agarose (Santa Cruz Biotechnology Inc, Santa Cruz, CA) was added, followed by another hour
of incubation. Immunocomplexes were washed twice in low salt buffer and
once with NET-gel buffer (50 mmol/L Tris-HCl, pH 7.5, 50 mmol/L NaCl,
0.1% NP-40, 0.2% gelatin) for 30 minutes at 4°C. Subsequently,
samples were prepared for SDS-PAGE and Western blotting. For
immunoprecipitation experiments using lysates derived from metabolically labeled cells, gels were treated with glacial acetic acid
and 20% 2,5-diphenyloxazole (Sigma) followed by drying and autoradiography. For coprecipitation experiments, in vitro translation yielding 35S-methionine-labeled FAA and FAC was performed
using the TNT T7 coupled reticulocyte lysate system (Promega, Madison,
WI) according to the manufacturer's instructions. GST-FAC bound
beads used in pull-down experiments have been
described.18 Potential complexes were allowed to form in
low salt lysis buffer under the same conditions as described above.
| |
RESULTS |
Generation and characterization of anti-FAA antibody.
We have generated a rabbit polyclonal antibody directed against the
amino-terminal portion of FAA expressed in bacteria. After affinity-purification, Western blotting experiments show that the
antibody detects the purified antigen His-FAA1 specifically (Fig 1A).
Western blots of extracts derived from lymphoblastoid cell lines show
that the antibody can also detect the 163-kD endogenous FAA protein
(Fig 1B). Slightly varying levels of FAA are found in cells derived
from normal controls or from patients representing FA complementation
groups B to E. By contrast, little if any FAA is detected in the FA-A
cell line HSC72, which was used previously for expression cloning of
the FAA cDNA and has been shown to lack the FAA mRNA.12
After transduction of HSC72 cells with a retroviral vector encoding the
FAA cDNA, FAA expression is restored (Fig 1B). In another FA-A cell
line, HSC99, FAA expression is normal, suggesting that as yet unknown
mutations in the two alleles of the FAA gene lead to the
expression of defective proteins. After stable transfection with an
episomal expression vector containing the FAA cDNA, higher levels of
FAA are detected (Fig 1B). As expected, restoration of wild-type FAA
expression in both cell lines corrects their sensitivity to MMC (data
not shown). FAA can also be immunoprecipitated from extracts of
metabolically labeled COS-1 cells that are transiently transfected with
a vector encoding FAA cDNA (Fig 1C). These findings show that the
anti-FAA antibody specifically detects FAA in different biochemical
assays.
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| Fig 1.
Generation of an affinity-purified rabbit polyclonal
antibody against FAA that specifically detects FAA in Western blotting
and immunoprecipitation experiments. (A) Western blot showing the
approximately 37-kD His-FAA1 fusion protein in bacterial extracts and
after purification using the affinity-purified antibody. The
His-tagged fusion protein encompasses the first 321 amino-terminal
residues of FAA and was used to raise the antibody. Lysates derived
from noninduced bacteria or isopropyl--thiogalactopyranoside
(IPTG)-induced bacteria are indicated with ( ) or (+),
respectively, and the purified protein with (P). (B) Western blot using
extracts derived from normal cells or cells representing five different
FA complementation groups. The FAA-specific band of approximately 163 kD is indicated. Blots were reprobed with an anti--tubulin antibody
as a control for the amount of protein loaded per lane. Lymphoblastoid
cell lines used represent the following FA subtypes: HSC93 and nl,
normal cells; HSC72 and HSC99, FA-A; HSC230, FA-B; HSC536, FA-C; HSC62,
FA-D; EUFA130, FA-E. HSC72+FAA indicates HSC72 cells stably
transduced with a retroviral vector expressing FAA. HSC99+FAA
indicate cells that were stably transfected with an episomal expression
vector containing the FAA cDNA. (C) Immunoprecipitation of FAA from
whole-cell extracts, generated in RIPA buffer, from metabolically
labeled COS1 cells either mock-transfected () or transiently
transfected with pcDNA-FAA5.5 (+).
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| Fig 2.
FAA is present in both the nuclear and the cytoplasmic
compartment. (A) (see page 2232) Costaining of FAA and IgG heavy
chain-tagged FAC in transiently transfected Hela cells by
immunofluorescence. (B) Western blotting on subcellular fractions
derived from 293 cells transiently expressing FAA. n, nuclear fraction;
c, cytosollic fraction; m, membrane fraction.
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Subcellular localization of FAA.
Next, we examined the subcellular localization of FAA in human cells by
immunofluorescence microscopy. Simultaneously, we also determined the
localization of FAC using a functionally active chimera, FAC1,
derived by fusion of FAC to the IgG1 heavy chain.18 In HeLa
cells transiently expressing FAA and FAC1, the large majority of
cells (approximately 90%) show FAA to be present in both the nuclear
and cytoplasmic compartment in varying ratios. In about 5% of
FAA-positive cells, FAA is detected predominantly in the nucleus and in
5% in the cytoplasm. Nuclear-localized FAA shows punctate forms in
occassional cells. Examples of different FAA staining patterns are
shown in Fig 2A. No staining is seen without
permeabilization, and FAA localization is similar in the absence or
presence of overexpressed FAC1 (data not shown). Consistent with
previous studies14,15 and similar to the expression of nontagged FAC, FAC1 is predominantly localized to the cytoplasmic compartment. Western blotting experiments using subcellular fractions derived from 293 cells transiently expressing FAA show that an estimated 20% of FAA is associated with intracellular membranes (Fig
2B), similar to that reported previously for FAC.15 Thus, the distribution of FAA is variable, which suggests that FAA shuttles between the cytoplasm and nucleus.
No evidence for an interaction between FAA and FAC.
The similarity in the clinical and cellular phenotypes of FA belonging
to different complementation groups has led to the suggestion that FA
proteins may participate in protein-protein interactions with each
other in macromolecular complexes.3,18 Here we have used
several strategies to show possible physical interactions between FAA
and FAC. First, we do not observe any interactions in coprecipitation
experiments using extracts from metabolically labeled 293 cells
transiently expressing FAA, FAC, or both
(Fig 3A). It should be noted that, under
similar conditions, FAC binds to the molecular chaperone
GRP9419 and to NADPH cytochrome P450 reductase (Kruyt FAE,
Hoshino T, Liu JM, Joseph P, Jaiswal AK, Youssoufian H, manuscript
submitted). Second, no FAC-FAA complexes are observed in
293 cells (Fig 3B) or in HeLa and COS-1 cells (data not shown) by
sequential immunoprecipitation and Western blotting. Third, mixing
35S-labeled FAA and FAC followed by immunoprecipitation
with anti-FAA or anti-FAC antibody in vitro (Fig 3C), or by pull-down
of 35S-labeled FAA with immobilized GST-FAC (data not
shown), reveal no interactions. These findings do not support the
existence of a macromolecular complex in which FAA and FAC
interact with each other, either directly or indirectly.
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| Fig 3.
FAA and FAC do not interact. (A)
Immunoprecipitation experiments using extracts derived from
metabolically labeled 293 cells that were transiently transfected with
the indicated vectors expressing FAA or FAC. Extracts were prepared in
RIPA buffer. (B) Immunoprecipitation of FAA and FAC using extracts
derived from 293 cells transiently expressing FAA, FAC, or FAA and FAC
as indicated. Extracts were prepared in medium salt buffer. Arrowheads
indicate the heavy and light chains of the antibodies. (C)
Coprecipitation experiments with in vitro-translated
35S-methionine-labeled FAA and FAC under low salt
conditions. Total reticulocyte lysate was loaded in lanes marked
FAA-lysate and FAC-lysate; control indicates total lysate without
template DNA. Full-length FAA and FAC are indicated.
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Nuclear localization of FAA is necessary and sufficient for
complementing MMC sensitivity.
Because FAA is present in both the cytoplasmic and nuclear
compartments, we asked whether a particular pool is necessary for protection against cross-linking agents. A previous attempt to address
this question by forced nuclear targeting of an FAA-GFP chimera was
inconclusive because of a lack of nuclear accumulation of the
chimera.17 In a reciprocal approach, we set out to generate a FAA-NES fusion protein to promote the export of nuclear FAA to the
cytoplasm. NES motifs have been shown to bind to Exportin1/CRM1 and
form complexes with the GTP-bound form of Ran.21,22
Subsequently, these complexes are transported through the nuclear pore
and dissociate rapidly upon arrival in the cytoplasm, accompanied by
the hydolysis of RanGTP to RanGDP.23 We fused the
well-characterized NES found in the inhibitor of cAMP-dependent protein
kinase24 to the carboxy terminus of FAA to generate
FAA-NES. As a control, we introduced an amino acid substitution that
renders the NES motif inactive24 to derive FAA-P12 (see
Fig 4A). Transient expression
of FAA-NES in HeLa cells and subsequent immunofluorescence microscopy
shows an apparent exclusive cytoplasmic localization (Fig 4B, see page 2232), whereas FAA-P12 transfected cells show both nuclear and cytoplasmic expression patterns similar to that of nontagged FAA (not
shown). In HSC72 cells stably expressing these constructs (Fig 4C),
FAA-NES fails to complement, while FAA-P12 is fully capable in
restoring MMC resistance (Fig 4D).
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| Fig 4.
Depletion of FAA from the nucleus abolishes its ability
to complement MMC hypersensitivity in FA-A lymphoblasts. (A) Schematic
representation of FAA-NES and FAA-P12. The double-headed arrow
indicates the amino acid substitution leading to inactivation of the
NES. (B) (see page 2232) Immunofluorescence microscopy using
pcDNAFAA-NES in transiently transfected Hela cells. (C) Western blot
showing the stable expression of FAA, FAA-NES, and FAA-P12 in HSC72
cells. (D) MMC sensitivity assays of the HSC72-derived stable
transfectants. ( ), Control; ( ), FAA; ( ), FAA-P12; ( ),
FAA-NES. A representative experiment is shown.
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The succesful use of the NES motif led us to reevaluate the effect of a
heterologous NLS motif on FAA localization and function. In a previous
study, the SV40 large T NLS motif was fused to the amino terminus of
FAA-GFP,17 but no change in the localization of FAA was
appreciated. In this study, the NLS motif was added to the carboxy
terminus of FAA (Fig 5A). As
a control, FAA was also fused to a previously described inactive NLS
motif.25 Upon expression in HeLa cells, strong nuclear
accumulation of FAA-NLS was seen readily by immunofluorescence, but no
staining in the cytoplasm could be detected (Fig 5B, see page
2232). FAA-mutNLS localization was similar to the nontagged FAA
(not shown). After stable expression in HSC72 (Fig 5C), FAA-NLS was
fully able to complement the hypersensitivity of these cells to MMC
(Fig 5D), as was FAA-mutNLS. These data show that expression of FAA in
the nucleus is essential for its cross-linker complementing function, while depletion of FAA from the nucleus abolishes this activity completely. Thus, the presence of FAA in the nucleus is both necessary and sufficient (ie, coexpression in the cytoplasm is not required) for
counteracting the cytotoxic effects of MMC.
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| Fig 5.
Nuclear-targeted FAA is fully functional in complementing
MMC sensitivity. (A) Schematic representation of FAA-NLS and
FAA-mutNLS. (B) (see page 2232) Visualization of FAA-NLS transiently
expressed in HeLa cells by immunofluorescence microscopy. (C) Western
blot showing the stable expression of FAA-NLS and FAAmutNLS in HSC72
cells. (D) MMC sensitivity assays of the HSC72-derived stable
transfectants. (), Control; (), FAA; (), FAA-NLS; (),
FAA-mutNLS. A representative experiment is shown.
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DISCUSSION |
In this study we present biochemical, cellular, and genetic data to
show that the two currently known FA proteins function in separate
subcellular compartments to protect cells against cytotoxicity by
cross-linking agents. To validate our conclusions, we have
characterized our affinity-purified polyclonal antibody using a number
of different strategies and addressed the issue of localization using
physical as well as functional endpoints. Overall, we believe that this
antibody is monospecific, as indicated by the single FAA-specific band
that is immunoprecipitated from COS-1 (Fig 1C) or 293 (Fig 3A and
B) cell extracts. However, although overexpressed FAA is readily
detectable by a variety of assays, immunodetection of endogenous FAA is
more challenging, presumably because of low expression levels. For
example, whereas endogenous FAA is clearly detectable by Western
analysis, detection by immunofluorescence has not been possible. Thus,
we have analyzed endogenous FAA by a more limited repertoire of
techniques. Nevertheless, comparative cell fractionation studies show
that there are no major differences between the localization of
endogenous and transfected FAA (not shown).
Our results show that FAC and FAA differ from each other by several
important criteria. For FAC, there is general agreement on the fact
that the protein resides predominantly in the
cytoplasm.14,15,26 Although Hoatlin et al26
recently reported that a small fraction (approximately 10%) of FAC in
transfected cells is nuclear, the physiological significance of this
observation remains unclear because cytoplasmic localization is
required for at least one major function of FAC: Fusion of a foreign
NLS to FAC directs the protein to the nucleus and abolishes its
complementation function in FA-C cells.16 In addition, the
mechanism by which FAC acts to protect against cross-linkers has been
at least partly elucidated by our recent finding that FAC binds to the
integral microsomal enzyme NADPH cytochrome-P450 reductase. This
binding attenuates the activity of the reductase and inhibits
conversion of the inert MMC to metabolically active forms (Kruyt FAE,
Hoshino T, Liu JM, Joseph P, Jaiswal AK, Youssoufian H, manuscript
submitted). FAC may function as a sensor in this major
cellular detoxification pathway.
For FAA, there is greater controversy with regard to its subcellular
localization. Kruyt et al17 showed that an FAA-GFP chimera
is localized primarily to the cytoplasm, and that mutation of the
putative NLS does not disturb its complementation function. It is
possible that fusion of the GFP epitope to the carboxy terminus interferes with FAA import into the nucleus, and a smaller, yet active,
nuclear fraction remains undetectable by this method. Similarly, the
lack of nuclear accumulation found with an NLS/FAA-GFP chimera may be
due to the GFP epitope. Alternatively, the NLS motif fused to the amino
terminus of FAA may be masked. Subsequently, Kupfer at al27
showed by using single method, ie, cell fractionation and
immunoprecipitation, that FAA is localized both in the nucleus and
cytoplasm. In addition, they also demonstrate that FAC is localized in
the nucleus with an estimated cytoplasmic/nuclear ratio of 3.2 in
extracts prepared from a population of cells, in contrast to their
previous reports showing FAC only in the cytoplasm.14,28 To
the extent that FAA is found in both the nucleus and cytoplasm, our
present results are in agreement with their data. By immunofluorescence
studies, we further show that the cytoplasmic/nuclear ratio of FAA can
differ in individual cells. This observation suggests that FAA shuttles
between the nucleus and cytoplasm. By using foreign nuclear export or
nuclear import signals, we have been able to dissect the contributions of each pool to the complementation function of FAA. Our results clearly show that localization of FAA to the nucleus is both necessary and sufficient for its ability to correct the cross-linker sensitivity of FA-A cells. A direct role for FAA in the nucleus may possibly involve DNA repair or recombination.
Unlike Kupfer et al,27 we find no complex formation between
FAA and FAC either in vivo or in vitro, and under varying experimental conditions and in different cell lines. It should be noted that these
assays have previously allowed us to identify a number of FAC
interacting proteins, and we have no obvious explanation for these
discrepancies. Although it is possible that the amino-terminal anti-FAA
antibody used in our study may block the domain(s) involved in protein
interactions and thereby prevent FAA-FAC complex formation, it seems
unlikely for several reasons. In contrast to monoclonal antibodies, it
would be highly unusual for polyclonal antibodies to function as
blocking antibodies. These antibodies recognize multiple epitopes, and
it would be reasonable to consider that at least some of the
epitopes are physically distinct from sites of protein-protein
interaction. Similar to Kupfer et al,27 we also used the
reciprocal approach of FAC immunoprecipitation followed by FAA
immunoblotting in the hope of further reducing the possibility of
epitope-shielding, but again we did not detect any interactions. Certainly, in our experience this approach has not presented any particular difficulties with the demonstration of interactions between
FAC and GRP9419 and between FAC and NADPH cytochrome p450
reductase (Kruyt FAE, Hoshino T, Liu JM, Joseph P, Jaiswal AK,
Youssoufian H: manuscript submitted). In addition, we
eliminated the antibody approach and instead used a functional,
epitope-tagged form of FAC, FAC1,18 but to no avail
(data not shown). Finally, we repeated the immunoprecipitation step
using either a polyclonal antibody against FAC or against the carboxy
terminus of FAA (both antibodies provided by Dr Alan
D'Andrea, Dana-Farber Cancer Institute, Boston, MA)27
followed by immunoblotting from control and FAA-complemented HSC72
lysates. We found no interaction between FAA and FAC (data not shown).
These results force us to conclude that there is no interaction between
FAC and FAA.
Although attractive as a hypothesis to explain the similarity in FA
pathogenesis, it is by no means obvious that FA proteins must
collaborate at some level to guard against cross-linker cytotoxicity. If, indeed, such collaboration exists, our results would be consistent with a model in which the two known FA proteins function in a linear
pathway, wherein FAC acts at a proximal step in the cytoplasm and FAA
at a more distal step in the nucleus to protect against cross-linker
cytotoxicity and chromosomal instability.
| |
FOOTNOTES |
Submitted April 23, 1998;
accepted July 9, 1998.
Supported by grants from the Fanconi Anemia Research Fund and the
National Institutes of Health (HL52138).
Address reprint requests to Hagop Youssoufian, MD, Department of
Molecular and Human Genetics, Baylor College of Medicine, One Baylor
Plaza, S840, Houston, TX 77030; e-mail: hagopy{at}bcm.tmc.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr Hans Joenje (Free University, Amsterdam, The Netherlands)
for kindly providing the FAA cDNA and the lymphoblastoid cell lines
representing the different FA complementation groups, Dr Christopher
Walsh (University of North Carolina, Chapel Hill) for providing the
retroviral vector-transduced HSC72 cell line, and Drs Phil Hastings and
Huda Zoghbi (Baylor College of Medicine, Houston, TX) for critical
comments on the manuscript and use of the fluorescence microscope,
respectively.
| |
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Abstract
Introduction
Abstract