Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 719-720
BRIEF REPORT
Strong FANCA/FANCG but weak FANCA/FANCC interaction in the
yeast 2-hybrid system
Tanja Reuter,
Sabine Herterich,
Oliver Bernhard,
Holger Hoehn, and
Hans J. Gross
From the Departments of Biochemistry and Human Genetics, University
of Würzburg, Würzburg, Germany.
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Abstract |
Three of at least 8 Fanconi anemia (FA) genes have been cloned
(FANCA, FANCC, FANCG), but their functions remain unknown. Using the
yeast 2-hybrid system and full-length cDNA, the authors found a strong
interaction between FANCA and FANCG proteins. They also obtained
evidence for a weak interaction between FANCA and FANCC. Neither FANCA
nor FANCC was found to interact with itself. These results support the
notion of a functional association between the FA gene products.
(Blood. 2000;95:719-720)
© 2000 by The American Society of Hematology.
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Introduction |
An important way in which gene function can be
elucidated is in the analysis of protein-protein interactions.
Intracellular distribution and putative interactions remain
controversial in Fanconi anemia (FA) gene products, as illustrated by
recent articles1,2 and by subsequent correspondence in this
journal.3,4 Immunoprecipitation data suggest that the FA
gene products interact in a multimeric protein complex.1,5
We applied the yeast 2-hybrid system6 to test independently
the concept of FA protein interaction.
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Materials and methods |
For constructing lexA fusions, FANCA, FANCC, and FANCG full-length
cDNA was subcloned to the plasmid pEG202. To generate B42 fusions, the
respective FA cDNA was subcloned to pJG4-5. These constructs were
transformed to the yeast strain EGY48 and tested for protein expression
by immunoblot analysis (data not shown). The control plasmid pRFMHI
encodes the Gal4 transactivator and serves as a positive control for
activation of the reporters. Similarly, the pSH17-4 plasmid encodes the
Drosophila bicoid protein, which does not activate the
reporters. Because the lexA-FANCG hybrid protein resulted in
transactivation of the reporters, FANCG could only be tested in fusions
with B42.
Yeast strain EGY48 was cotransfected in pairs with different
combinations of vectors expressing the full-length FA lexA or B42
fusion protein. Positive interaction was documented by the ability of
yeast cells to grow on galactose medium, assayed by the expression of
the chromosomal leucine reporter and by episomal LacZ expression.
Reduced growth on glucose minus leucine, or on glucose medium
containing X-Gal (compared to the corresponding galactose plates)
served as controls for an interaction because expression of the B42
fusion protein is repressed on glucose plates. The percentage of yeast
recombinants that may generate false-positive results by activation of
the reporter genes was less than 4%.
| |
Results and discussion |
The FANCA protein interacted strongly with FANCG, as evidenced by
LEU2 and LacZ expression (Figure 1).
Activation of the LEU2 reporter gene was also observed when lexA-FANCA
was cotransfected with B42-FANCC. However, this interaction was much
weaker and could not be detected by the less sensitive LacZ reporter
assay (data not shown). There was no evidence for interaction using the
inverse combination. There also was no detectable interaction between
the FANCC and FANCG gene products, nor was there evidence that FANCA
and FANCC interact with themselves. Marginal growth of the FANCA/FANCA
cotransformants on leucine-deficient galactose plates did not result
from interaction because such cotransformants spotted onto glucose
medium and lacking leucine behaved like those spotted onto galactose
plates. In contrast, there was a consistent difference between the
growth performance of the FANCA/FANCC cotransformants, depending on
whether they were plated onto leucine-free glucose or galactose medium
(Figure 1). To determine whether a mutant FANCC protein could abolish
the FANCA/FANCC interaction, we generated L554P-FANCC mutant fusion
constructs. These mutant hybrid proteins did not enter the yeast
nucleus reliably, so these interaction assays were not informative. In
the liquid beta-galactosidase assay, activation of LacZ by the
FANCA/FANCG interaction was nearly as efficient as activation by the
natural yeast transcriptional activator Gal4, confirming a very strong
interaction. Quantification of activation by all other cotransformants
resulted in beta-galactosidase activities of less than 5% of the
FANCA/FANCG interaction (data not shown). Given the permissiveness of
the yeast 2-hybrid system, the relatively weak interaction between
FANCA and FANCC could also be a consequence of transcriptional
activation by spurious hydrophobic interactions. In addition, the
detection of minor interactions may critically depend on the choice of
fusion partners and on other technical features of the 2-hybrid
system.7 However, the likely importance of FANCA as a core
protein in a multimeric complex is strongly supported by the prominent
FANCA/FANCG interaction.5
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| Fig 1.
Yeast plates showing growth response under different
dilution and testing conditions.
Yeast strain EGY48 was cotransfected in pairs, with the indicated
combinations of vectors for the expression of full-length Fanconi
anemia LexA or B42 fusion proteins. Three dilutions each were spotted
onto complete minimal dropout media. The first protein of each of the
combinations represents the LexA fusion, the second protein the B42
fusion. Each result was confirmed by 4 independent assays.
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Acknowledgment |
The authors thank Hans Joenje (Free University, Amsterdam, The
Netherlands) for providing the FANCA and FANCC cDNA.
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Footnotes |
Submitted May 10, 1999; accepted September 15, 1999.
Supported by the Schroeder-Kurth Fonds, Germany.
Reprints: Tanja Reuter, Department of Biochemistry,
University of Würzburg, Biozentrum, D-97074 Würzburg,
Germany; email: reuter{at}biozentrum.uni-wuerzburg.de.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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References |
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Naf D, Kupfer GM, Suliman A, Lambert K, D'Andrea AD.
Functional activity of the Fanconi anemia protein FAA requires FAC binding and nuclear localization.
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The Fanconi anemia proteins FAA and FAC function in different cellular compartments to protect against cross-linking agent cytotoxicity.
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3.
Garcia-Higuera I, D'Andrea AD.
Regulated binding of the Fanconi anemia proteins, FANCA and FANCC.
Blood.
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4.
Kruyt FAE, Youssoufian H.
Response.
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5.
Garcia-Higuera I, Kuang Y, Naf D, Wasik J, D'Andrea AD.
Fanconi anemia proteins FANCA, FANCC and FANCG/XRCC9 interact in a functional nuclear complex.
Mol Cell Biol.
1999;19:4866[Abstract/Free Full Text].
6.
Fields S, Song O.
A novel genetic system to detect protein-protein interactions.
Nature.
1989;40:245.
7.
Mathew CG, Morgan N, Tipping A, Huber P.
Molecular pathology and functional analysis in Fanconi anemia [abstract].
Am J Hum Genet.
1998;63:A373.
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Abstract
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