From the INSERM U410, Faculté de Médecine
X. Bichat, 16 Rue Henri Huchard, 75018 Paris, France and the
¶ Faculté Agronomique de Gembloux, Centre de Biophysique
Moléculaire Numérique, 2 Passage des Déportés,
5030 Gembloux, Belgium
Received for publication, November 2, 2000
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ABSTRACT |
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We screened a cDNA bank of rabbit gastric
fundic mucosa by two-hybrid assays looking for binding partners of the
N-terminal domain of the rabbit gastric H,K-ATPase. We extracted
five clones sharing more than 90% sequence identity. The longest clone
codes for a protein sharing a high identity (96 and 96.8%,
respectively) with a fragment of the membrane domain, from
Arg-835 to Ser-873, plus the major part of the "spectrin
binding domain" going from Glu-874 to Leu-1455 of human and mouse
ankyrin III. We conclude that the membrane and spectrin binding domains
of the rabbit ankyrin III are candidates for the binding partner of the
N-terminal domain of the rabbit gastric H,K-ATPase. To validate the
ankyrin-ATPase interaction and to test its specificity, we produced
both domains in yeast and bacteria, coimmunoprecipitated them with an
anti-ATPase antibody, and copurified them by affinity chromatography.
The sequence of rabbit ankyrin III was not known, and this is the first
report demonstrating that the ankyrin III and the H,K-ATPase interact
with no intermediate. The interaction involves the N-terminal domain of
the ATPase on one hand and the spectrin binding domain of the
ankyrin on the other.
The sequestration of molecules in intracellular compartments is a
fundamental capacity of all living cells, and in polarized cells, the
sequestration is directional. For instance, the plasma membrane that
envelops all epithelial cells has two domains, basal and apical. Both
correspond to different components. Membrane proteins must be targeted
to one or the other domain after synthesis. The mechanisms of targeting
are not yet fully understood, but the cell skeleton has a major role,
at least by leading proteins to, and holding them in, their membrane
domains (1-3).
Linkers are required for the interaction of some membrane proteins such
as AE1 (anion exchange 1 or band 3 protein) and Na,K-ATPase with
skeleton components. The AE1 and Na,K-ATPase linker is ankyrin (4-6).
Smith et al. (7) previously demonstrated the colocalization of ankyrin and H,K-ATPase in parietal cells and the
coimmunoprecipitation of the gastric pump with exogenous erythrocyte
ankyrin. This supports the idea that the gastric ATPase can bind
ankyrin as its parent protein, the Na,K-ATPase, does. However, it is
not known how ATPase and ankyrin interact and whether minor cellular
components act as linkers.
In this study, we demonstrate that the rabbit gastric H,K-ATPase
binds to ankyrin III. We map the binding site in the N-terminal (Nt)1 domain of ATPase on one
hand and in the spectrin binding domain of ankyrin III on the other
hand. The spectrin binding domain of the rabbit ankyrin III is cloned
and has more than 90% identity with the analogous domains of the mouse
and human ankyrin. Fragments of rabbit ATPase and rabbit ankyrin III
were both expressed to test the specificity of the interaction by
coimmunoprecipitation and affinity chromatography.
Library Construction--
Stomachs were collected from
Nembutal-injected rabbits. Gastric fundic mucosa was scrapped and
immediately frozen in liquid nitrogen. Samples were sent to
CLONTECH Laboratories, Inc. for preparation of the
cDNA bank with (dT) and random oligomers to limit the
overexpression of 3' extremities of mRNA.
cDNA were ligated in the prey plasmid pGAD 10 using
EcoRI adaptors and incorporated in DH5 Construction of the Bait Plasmid--
The DNA fragment for the
first 98 residues of the rabbit gastric H,K-ATPase was obtained by PCR
from the complete cDNA. The PCR conditions were as follows: (i)
denaturation at 95 °C for 10 min; (ii) 35 cycles each consisting of
denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min,
and elongation at 72 °C for 30 s using Vent polymerase (New
England Biolabs); and (iii) a final elongation at 72 °C for 10 min.
The PCR product with the expected size of 320 base pairs was digested
with BamHI and SalI and purified on a 4% agarose
gel with a QIAquick gel extraction kit (Qiagen) according to the
manufacturer's instructions. It was inserted in the
BamHI/SalI site of the pFBL23 plasmid (a gift of
J. Camonis, Institut Curie, Paris, France), and the product was
sequenced (Genome Express, Grenoble, France).
Library Screening--
Yeast strain HF7c (a donation of J. Camonis, Institut Curie, Paris, France) was transformed to Trp
prototrophy with pFBL23-Nt using the lithium acetate method (8). The
resulting strain was further transformed to Trp, Leu, His prototrophy
with the pGAD10 library plasmid. The transformation mix (100 µl) was
plated on a 10-cm Petri dish and incubated at 30 °C for 5 days.
Aliquots of each mix were taken before plating and used to determine
the transformation efficiency on Trp- and Leu-free medium.
His+ colonies were screened for
Plasmids were extracted and recovered by electroporation in the
leu Coimmunoprecipitation of the Two-hybrid Proteins Expressed from
Yeast--
Yeast extracts were prepared from liquid cultures in
SD-Trp-Leu-His medium (CLONTECH
Laboratories, Inc.). Cultures were grown to an absorbance at 600 nm of
0.8. The yeast extracts were centrifuged (5,000 × g
for 5 min), and the pellets were washed in 50 mM
Na2HPO4, 300 mM NaCl, pH 8. The
yeast extracts were lysed by vortexing with glass beads (reference
number 854 170/1, Braun Biotech International). Cell debris was
discarded by centrifugation (20,000 × g for 20 min),
and the supernatants were incubated for 4 h at 4 °C in the presence of 10 µg of the anti-Nt H,K-ATPase antibody (95A3). Then, 10 µg of IgG2 against mouse IgG were added and incubated for 4 h
at 4 °C. Immune complexes were collected on protein A-Sepharose (Amersham Pharmacia Biotech), separated by SDS-PAGE, and blotted onto nitrocellulose.
Construction of Vectors Expressing the Nt of H,K-ATPase--
The
DNA sequence encoding for the Nt (amino acids 1-98) was obtained from
the complete cDNA of rabbit gastric H,K-ATPase cloned in Bluescript
vector by PCR amplification. The PCR conditions were the same as for
the bait plasmid. The final product of 320 base pairs was digested with
NcoI and BglII and purified on a 4% agarose gel
with a QIAquick gel extraction kit (Qiagen) using the manufacturer's
instructions. This cDNA was inserted between the restriction sites
NcoI and BglII of the prokaryotic expression vector pQE 60 (Qiagen) to generate a plasmid encoding for an in-frame fusion protein composed of Nt and 6 histidines in the C-terminal extremity. The plasmid with the insert was checked by sequencing (Genome Express).
Expression of the Nt of H,K-ATPase--
JS 238 E. coli strain (a gift of O. Fayet, Laboratoire de Microbiologie et
Génétique Moléculaire, Toulouse, France)
overexpressing the lactose operon repressor (Lac lq mutation) was
transformed by the CaCl2 method with pQE 60 containing the
Nt of the ATPase. Cells were grown at 37 °C in LB medium containing
100 µg of ampicillin until the absorbance at 600 nm reached 0.8 units. Expression of the fusion protein was induced by adding 1 mM isopropyl-1-thio- Construction of the Vector Expressing the Spectrin Binding
Domain--
The DNA sequence encoding for the spectrin binding domain
(SBD) was obtained by the PCR amplification of a clone isolated by the
two-hybrid screening. The PCR conditions were as follows: (i)
denaturation at 95 °C for 10 min; (ii) 35 cycles each consisting of
denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min, and elongation at 72 °C for 2 min with the Pfu polymerase
(reference number 60025251, Stratagene); and (iii) a final
elongation at 72 °C for 10 min.
The PCR product of 1850 base pairs was digested with BamHI
and Bsu36I and purified on a 1% agarose gel with a QIAquick
gel extraction kit (Qiagen) using the manufacturer's protocol. Then the full cDNA was inserted between the
BamHI/Bsu36I restriction sites of the yeast
expression vector pYeF2 2A (a gift of Center Génétique
Moléculaire, CNRS, Gif sur Yvette, France) to generate a
plasmid encoding for the SBD under the dependence of a
galactose-inducible promoter (pGAL10-CYC1) with an adenine selection
marker. The plasmid with insert was sequenced (Genome Express).
Expression of the Spectrin Binding Domain--
Expression was
performed in yeast strain BMA 64 (a donation of Center
Génétique Moléculaire, CNRS, Gif sur Yvette, France). Yeast were grown at 30 °C overnight in a 100-ml flask containing 25 ml of WO medium (yeast nitrogen base (Life Technologies, Inc. 25685-033), 1.43 g/liter; ammonium sulfate, 5 g/liter; galactose, 20 g/liter) complemented with 20 µg/ml uracil, 20 µg/ml tryptophan, 100 µg/ml leucine, and 10 µg/ml histidine. The following day, 250 ml of the same medium were seeded and incubated at 30 °C up to the absorbance at 600 nm of 0.8. Yeast were pelleted by
centrifugation (5,000 × g for 10 min), suspended in
2 ml of sonication buffer with 5 µl of protease inhibitor mixture (P
8215, Sigma), and then broken by vigorous vortexing with glass beads
(854 170/1, Braun Biotech International). Cell debris were discarded by
centrifugation at 4 °C (15,000 × g for 10 min).
Immunoprecipitation--
Four mixtures were prepared and
incubated at 4 °C for 4 h. These mixtures were as follows: (i)
100 µl of bacterial extract containing the Nt plus 2 ml of yeast
extract containing the SBD; (ii) 100 µl of bacterial extract
containing the Nt plus 2 ml of yeast extract without the SBD; (iii) 100 µl of bacterial extract without the Nt plus 2 ml of yeast extract
containing the SBD; and (iv) 100 µl of bacterial extract without the
Nt plus 2 ml of yeast extract without the SBD.
Ten µg of 95A3 were added and incubated for 4 h at 4 °C.
Then, 10 µg of IgG2 anti-mouse IgG were added for 4 h at
4 °C. Immune complexes were collected on protein A-Sepharose
(Amersham Pharmacia Biotech), separated by SDS-PAGE, blotted onto
nitrocellulose by electro-elution, and analyzed.
Affinity Chromatography--
Four columns of 500 µl of
nickel resins (superflow, Qiagen) were prepared and equilibrated
with 50 mM Na2HPO4, 300 mM NaCl, pH 8. In columns A and B, 10 ml of the bacterial
extract containing the Nt domain of the ATPase were poured. In C and D,
10 ml of the bacterial extract free of the Nt domain of ATPase were
poured. All columns were washed according to the manufacturer's
protocol. Three liters of a yeast culture containing the SBD fragment
of ankyrin were poured in columns A and C, and 3 liters of the same culture medium free of SBD were poured in columns B and D. All columns
were rinsed with 50 mM Na2HPO4, 300 mM NaCl, pH 8 and eluted with 500 mM imidazole.
The four eluates were analyzed by SDS-PAGE and Western blots.
Western Blots--
Nitrocellulose sheets were incubated in 1×
phosphate-buffered saline, 5% bovine serum albumin, 0.1% Tween 20 in
the presence of antibodies, either the 95A3 mouse IgG monoclonal
antibody against the Nt of the H,K-ATPase or the rabbit IgG polyclonal
antibody against the SBD of ankyrin III (a gift of K. Kordeli,
Laboratoire de Biologie Cellulaire, Institut J. Monod, Faculté de
Jussieu, Paris, France). For staining, secondary species antibodies
conjugated with horseradish peroxidase were used, and the stain was
revealed with the Pierce chemiluminescence kit (reference number
34080, Pierce) using the manufacturer's protocol.
Screening of the Gastric cDNA Bank
Seven million gastric cDNA clones were screened against the Nt
fragment of the H,K-ATPase by the two-hybrid assays; 7 million is
four times the amount of independent clones of the cDNA bank and
should thus be statistically sufficient to screen all the different
cDNA fragments. Selected were 144 positive clones, which amount to
20 positive clones per million. Five clones had more than 90%
identity. Three were identical (1,791 base pairs) and corresponded to 597 aa. The two others were shorter and overlapped the
previous sequence on 498 aa (1,494 base pairs). Those fragments have a
high identity (>96%) with the "membrane domain" of the human and
mouse ankyrin III from Arg-835 to Ser-873 and with the major part of
the spectrin binding domain going from Glu-874 to Leu-1455 (9).
By looking for a protein interacting with the N-terminal domain of the
rabbit gastric H,K-ATPase, we selected the membrane and spectrin
binding domains of the rabbit ankyrin III. The sequence shows little
variation between the human and mouse ankyrin III sequences (Fig.
1).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Escherichia
coli strain by efficiency transformation. The library had about
1.7 million independent clones, of which 30% were empty.
-galactosidase activity using the
filter assay method. Positively transformed colonies eliciting 50-unit
absorbance at 600 nm were blotted onto Whatman 3MM paper. The
filter was frozen in liquid nitrogen and thawed at room temperature. The freeze-thaw cycle was repeated three times. The filter was soaked
in 60 mM Na2HPO4, 40 mM
NaH2PO4, 10 mM KCl, and 1 mM MgCl2 containing 2 mg/ml
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside (X-gal)
and then incubated overnight at 37 °C. The clones expressing the
galactosidase were selected only when the Lex A DNA binding domain-Nt fusion was present.
E. coli strain 1066 (a gift of C. Cullin,
Center Génétique Moléculaire, CNRS, Gif sur Yvette,
France). The plasmids were then used to transform the HF7c strain alone
or with pFBL23 in the presence of Nt or a control protein (laminin,
hydrophobic protein). The clones expressing the Nt of H,K-ATPase were
selected when they were positive for Trp, Leu, His prototrophy and
-galactosidase activity. The plasmids were sequenced (Genome Express).
-D-galactopyranoside for
4 h at 30 °C. Bacteria were harvested at 4 °C by
centrifugation at 5,000 × g for 20 min. Pellets were
suspended in 50 mM Na2HPO4, 300 mM NaCl, pH 8 in the presence of the protease inhibitor
mixture (P 8215, Sigma). Bacteria were lysed by sonication (14 µm for 1 min) and centrifuged at 20,000 × g for 20 min at
4 °C. The supernatant was used for immunoprecipitations and affinity chromatographies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Alignment of the rabbit sequence with the
sequences of the SBD of human and mouse ankyrin III. The rabbit
sequence is the reference, and the nonidentical residues are in
gray boxes. H. sapiens, Homo
sapiens.
Characterization of the ATPase-Ankyrin Interaction
Coimmunoprecipitation of Fusion Proteins--
To check whether the
activation of the reporter genes of the two-hybrid assay is the
consequence of a specific interaction between the N-terminal domain of
the ATPase and the ankyrin fragment, we first tested whether the Nt
domain (expressed as Lex A DNA binding domain-Nt) and the SBD
domain (expressed as SBD-gal4-activating domain) can coprecipitate when
they are incubated in the presence of a specific anti-ATPase antibody.
Both proteins are recovered in the same precipitate (Fig.
2, lanes 1 and 4).
This precipitate is absent when the extracts of the bait Lex A DNA
binding domain-Nt cotransfected with gal4-activating domain (SBD-free)
and of the prey SBD-gal4-activating domain cotransfected with Lex A DNA
binding domain (Nt of ATPase-free) are used. This absence indicates
that the immunoreaction is specific. Thus the activation of the
reporter gene of the two-hybrid assays is due to the direct interaction of the Nt domain of the gastric ATPase with the ankyrin fragment.
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Coimmunoprecipitation of the Isolated Domains--
To corroborate
the previous conclusions, both proteins were expressed in the absence
of their fusion partners: the activating and binding domains. The SBD
(597 aa) was expressed in yeast, and the Nt of the ATPase (98 aa) was
expressed in E. coli. After mixing the yeast and E. coli extracts, the Nt anti-ATPase antibody (95A3) was added.
Precipitation was induced as described under "Experimental
Procedures." The SBD of ankyrin was recovered in the ATPase
precipitate (Fig. 3, lanes 1 and 2). If the ATPase is absent, the ankyrin fragment is not
precipitated, which implicates that the coprecipitation requires a
specific interaction (Fig. 3, lanes 3-6).
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Affinity Chromatography--
To further check the specificity of
the interaction, we tested whether the Nt-SBD complex was
recovered by affinity chromatography on nickel resin (Fig.
4). In two columns (A and B), we poured the bacterial extract containing the Nt fragment of the ATPase tagged
with 6 histidines (Fig. 4, a and b, lane
4). In two other columns (C and D) we poured the bacterial extract
free of the ATPase fragment (Fig. 4c, lane 4;
result not shown for column D, because no protein was eluted from this
column). Then, different yeast extracts were added. Those containing
the ankyrin fragment (Fig. 4, a and c, lane
3) were loaded on columns A and C, and those free of the ankyrin
fragment (Fig. 4b, lane 3 and unshown result
(column D)) were loaded on columns B and D.
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All columns were then rinsed and eluted. The presence of the Nt fragment of the ATPase and of the SBD fragment of ankyrin in the elution was demonstrated by SDS-PAGE and by Western blots. The fractions eluted from column A contain both proteins (Fig. 4a, lanes 1 and 2). Similar bands are absent from the elution of column C (Fig. 4c, lanes 1 and 2) and of column D (result not shown). Therefore, the Nt domain of the ATPase binds to the nickel resin and is able to hold back the SBD fragment. Moreover, the Western blot of the fractions loaded on, and eluted from, columns B, C, and D (Fig. 4, b and c; results from column D are not shown) argue for the specificity of the interaction because, in the absence of the Nt, of the SBD, or of both fragments, no immunoreactive protein is held back on the columns, as revealed by the antibodies.
Therefore, we conclude that the interaction between ATPase and ankyrin
fragments is specific. The Nt domain of the gastric ATPase and the SBD
of rabbit ankyrin III are responsible for this interaction. Because the
Nt and SBD fragments are the only proteins recovered by affinity
chromatography, we further conclude that the interaction is direct and
does not require a third partner (Fig.
5).
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DISCUSSION |
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Looking for cytoplasm partners of the N-terminal domain of the rabbit gastric H,K-ATPase, we screened a cDNA bank of rabbit fundic mucosa by two-hybrid assays. We identified the SBD of rabbit ankyrin III. Previous studies strongly suggested an interaction between the H,K-ATPase and an ankyrin. Smith et al. (7) demonstrated by indirect immunofluorescence and by confocal microscopy on isolated gastric glands that the H,K-ATPase colocalized with ankyrin in resting and stimulated parietal cells. In the resting cells, the distribution of both proteins is diffuse in the cytoplasm, whereas after stimulation, they are both located at the apical membrane. In the same work, Smith et al. (7) demonstrated that exogenous erythrocyte ankyrin coimmunoprecipitates with the parietal cell microsomes. Because the main protein of gastric microsomes is the proton pump, this H,K-ATPase was a good candidate for an interaction with ankyrin.
In this work we make the first direct demonstration that the ankyrin
III and the H,K-ATPase interact and that their interaction does not
require a third partner. Moreover, we demonstrate that the 80 residues
of the N terminus of the subunit of the gastric H,K-ATPase bind to
a fragment of ankyrin that contains the spectrin binding domain of ankyrin.
It has been demonstrated that the Na,K-ATPase and ankyrin
interact by way of several sites (10). The ATPase sites were mapped on
the second (aa 140-290) and catalytic (aa 345-784) loops of the subunit. It was further demonstrated that the ATPase interacts with the
repeat domain (89 kDa) and the SBD of ankyrin (10). The existence of at
least two sites explains the high affinity (Kd, 25 nM) of the complex, and the destruction of one site
decreases the affinity constant by 20-fold (5).
The gastric H,K-ATPase and the Na,K-ATPase are two homologous ionic pumps. They have a similar structure, with two subunits sharing a high degree of sequence identity (60-65% on the whole sequence). Homology is high, especially in the domains carrying the ankyrin Na,K-ATPase interaction sites. Therefore, the gastric H,K-ATPase could have ankyrin binding sites on the second loop (aa 155-300) and/or the catalytic domain (aa 357-777). Indeed, the catalytic domain of the H,K-ATPase has a 19-residue fragment conserved in the Na,K-ATPase and in the band 3 protein also. This fragment has been identified as an ankyrin binding site (11).
By two-hybrid assays, we tested whether loop II and the catalytic domain of the gastric ATPase interact with the SBD of ankyrin III. The ATPase fragments (residues Gln-161 to Thr-293 for loop II and Thr-357 to Arg-777 for the catalytic loop) failed to interact with the SBD. Two explanations can be offered. Either none of those H,K-ATPase domains binds to the SBD of ankyrin III, or the two-hybrid assays are inefficient. The latter hypothesis cannot be ruled out because, if the N-terminal of the ATPase is short and polar and thus has a good probability to be expressed as a native fold, loop II and the catalytic domain are longer and more hydrophobic. The expression of hydrophobic fragments often leads to inclusion bodies because the native fold is not reached.2 Lastly, it could be argued that this part of the H,K-ATPase binds to other domains of ankyrin (repeat domain, regulatory domain) that we did not use in our assays.
The N-terminal domain of the H,K-ATPase and the spectrin binding domain
of ankyrin interact. This interaction is specific to the gastric pump,
because the N terminus of the Na,K-ATPase does not interact.
Actually, no interaction of the N terminus of the Na,K-ATPase was
detected when the whole ankyrin III was used. The N-terminal fragments
of both ionic pumps are the most different parts of their sequences.
This could indicate different functions. The N terminus of the
H,K-ATPase could be a regulating element in the activation of parietal
cells by acting in the translocation of the pump to the apical
membrane. Indeed, this phenomenon is specific to the H,K-ATPase. It
relies upon the recruitment of the gastric parietal cell skeleton by as
yet unclear mechanisms. Ankyrin III could be a piece of the puzzle.
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ACKNOWLEDGEMENTS |
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We thank J. Camonis (Institut Curie, Paris, France), C. Cullin and J. M. Rouillard (Center Génétique Moléculaire, CNRS, France), O. Fayet (CNRS, Toulouse, France), and K. Kordeli (Institut J. Monod, Faculté de Jussieu, Paris, France) for donation of biological materials.
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FOOTNOTES |
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* This work was supported by INSERM and the National Funds for Scientific Research of Belgium.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by the Interuniversity Poles of Attraction Program, Belgian State Prime Minister's Office, Federal Office for Scientific, Technical, and Cultural Affairs (PAI Contract Number P4/03).
Research Director at the National Funds for Scientific
Research of Belgium (FNRS).
** To whom correspondence should be addressed: CBMN, 2 Passage des Déportés, 5030 Gembloux, Belgium. Tel.: 32 1 81 622 534; Fax: 32 1 81 622 522; E-mail: thomas.a@fsagx.ac.be.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M009980200
2 F. Festy, J.-C. Robert, R. Brasseur, and A. Thomas, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: Nt, N-terminal or N terminus; PCR, polymerase chain reaction; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; SBD, spectrin binding domain; aa, amino acids.
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REFERENCES |
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