From the Institut für Molekulare Genetik, Im
Neuenheimer Feld 230 and ¶ Biochemie-Zentrum Heidelberg,
Biologische Chemie, Im Neuenheimer Feld 501, Universität
Heidelberg, D-69120 Heidelberg, Germany
Received for publication, August 2, 2000, and in revised form, November 21, 2000
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ABSTRACT |
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Several unrelated proteins are known that
specifically interact with members of the mammalian hsp70 chaperone
protein family independent of the hsp70 substrate-binding site. One of
these is Hap46, also called BAG-1, which binds to the ATP-binding
domain of hsp70 and its constitutively expressed, highly homologous
counterpart hsc70, thereby affecting nucleotide binding, as well as
protein folding properties, of these molecular chaperones. In an
attempt to delineate the potential contact sites on hsp70/hsc70
involved in this interaction we made use of the following two
independent approaches: (i) screening of membrane-bound peptide
libraries based on the sequence of the ATP-binding domain and (ii) the
phage-display technique with random dodecapeptides. These approaches
yielded partially overlapping results and identified several possible contact regions. On the space-filling model of hsc70, the two major
contact areas for Hap46 delineated in the present study are located on
the same side of the molecule on either subdomain that border the
central cleft harboring the nucleotide-binding site. We suggest that
this bridging affects the conformation of the ATP-binding domain in a
way similar to the opening of the nucleotide-binding cleft produced in
the bacterial hsp70 homologue DnaK upon binding its regulatory protein GrpE.
Members of the hsp70 (70-kDa heat
shock protein) family are among the most
abundant soluble proteins in mammalian cells, most notably the
constitutively expressed form, hsc70 (for reviews see Refs. 1-3). They
are involved in a great variety of functions, i.e. they prevent hydrophobic areas of proteins
from aggregating, function in the folding and unfolding of protein
structures, participate in transport into cellular organelles (4, 5),
deliver proteins for intracellular degradation (6), and play a role in
the survival of cancer cells (7). Often the activities of hsp70s, for
example in protein folding reactions, are stimulated by hsp40 or other members of the DnaJ-like protein family (8). Common to all hsp70
proteins is a highly conserved domain structure; the 44-kDa ATP-binding
domain occupies more than the amino-terminal half of the molecule and
is made up of two subdomains of about equal size and similar
three-dimensional structure (9-11). Substrate binding employs the
carboxyl-terminal part of the molecule that is composed of two very
differently structured subdomains (12). One of the most interesting
open questions at present is how these major hsp70 domains interact
with each other to bring about the biological effects of hsp70
chaperones. Such interdomain interactions appear particularly relevant
in situations when various accessory proteins are present that upon
association modulate the functions of hsp70 molecular chaperones.
In addition to cochaperones of the DnaJ/hsp40 family, several unrelated
protein factors have been identified that specifically bind to
mammalian hsp70s. The hsc70-interacting protein Hip, also called p48,
was the first (13, 14). The hsp70/hsc70-associating protein Hap46, also
known as BAG-1, has originally been detected by interaction screening
approaches using as bait a nuclear receptor, the anti-apoptotic protein
Bcl-2, or a membrane receptor of the tyrosine kinase family (15-17)
but was subsequently found to directly interact with members of the
hsp70 family (18-21). Third is the hsp70-interacting protein HspBP1
(22, 23). All these factors are known to interact with the ATP-binding
domain of hsp70 chaperones and have been found to affect their
biochemical activities, albeit in different ways. By contrast, the
carboxyl-terminal domain of hsp70s is involved in interactions with the
hsp70/hsp90-organizing protein Hop/p60 (18, 24, 25) and the carboxyl
terminus of Hsc70-interacting protein CHIP (26). Nevertheless,
DnaJ/hsp40 and CHIP affect the ATPase activity of hsp70s even though
they bind to the distally located carboxyl-terminal part (18, 25, 26).
Moreover, the interaction of Hop/p60 is inhibited when Hap46 binds to
the amino-terminal domain of hsc70 (24). These observations suggest
that there are intricate interrelationships between hsp70 domains and
the proteins associating with them.
In an attempt to achieve a better understanding of the molecular
interactions between hsp70s and their accessory proteins we
investigated the ATP-binding domain of mammalian hsc70 for contact
sites with Hap46. To this end, we employed as complementary approaches
(i) screening of membrane-bound peptides covering the sequence of the
ATP-binding domain of hsc70 and (ii) the phage-display technique using
random peptide libraries. Several potential contact sites were detected
by these independent techniques. Our data suggest that Hap46 binds to
hsc70 from one side of the ATP-binding domain by making use of two
major contact areas.
Expression of Recombinant Hap46--
The fusion protein of
glutathione S-transferase with Hap46 was expressed in
Escherichia coli JM109 and purified on GSH-Sepharose as described earlier (15). Cleavage was with thrombin (27).
Synthesis of Immobilized Peptides and
Immunodetection--
Peptides were synthesized on activated membranes
containing a polyethylene glycol 600 amino spacer (Abimed) using
Fmoc chemistry (28). After completion of the synthesis, membranes were
washed three times with phosphate-buffered saline and blocked overnight in saline containing 5% dried milk powder. Following a wash with saline containing 0.2% Tween 20, membranes were allowed to react over
night with 1-2 µg/ml Hap46 in saline containing milk powder. Unbound
Hap46 was removed by three washes in saline containing 0.2% Tween 20 whereupon membranes were incubated for 4 h with appropriate
dilutions of monoclonal antibodies CC9E8 (29) and 3.10G3E2 (30). Bound
Hap46·antibody complexes were visualized using a 1:5000 dilution of
horseradish peroxidase-coupled goat anti-mouse antibody (Dianova) in
saline containing milk powder for 1 h, followed by detection via
enhanced chemoluminescence (ECL; Amersham Pharmacia Biotech).
In an attempt to reuse membranes pre-exposed to Hap46 and antibodies,
the membranes were stripped by 3 washes each in the following
solutions: (i) dimethylformamide, (ii) 8 M urea, 10% SDS,
1% Epitopes on Hap46 Recognized by Monoclonal Antibodies CC9E8 and
3.10G3E2--
Epitopes were mapped by screening of membrane-bound
pentadecapeptides encompassing the complete amino acid sequence of 274 residues of human Hap46 (15), staggered by two amino acids. Peptide
synthesis and antibody reactions were performed as described above.
Antibody 3.10G3E2 recognized the amino acid sequence RSEEVTREEMA, corresponding to amino acids 59-69 of Hap46, whereas antibody CC9E8
reacted with amino acids 151-161, encompassing the sequence NSPQEEVELKK.
Phage Display--
Pannings were carried out essentially as
described before (33, 34) with 15-20 µg of recombinant Hap46
immobilized on Petri dishes (3.5-cm diameter; Greiner), blocked, and
incubated with 5 × 109 phage particles from a 12-mer
random peptide phage-display library (PhD library; New England
Biolabs), following the manufacturer's protocol. To eliminate
enrichment of phage clones interacting with the plastic surface or the
blocking agent, the following two strategies were employed: (i) eluted
and amplified material from the first panning was either subjected to a
preadsorption step on bovine serum albumin-coated Petri dishes
prior to subsequent pannings, or (ii) bovine serum albumin as blocking
agent was alternated with casein in the form of dried milk powder.
After the second and third round of panning, phage clones were picked
at random and amplified individually. Following standard procedures,
DNA was purified and subjected to cycle sequencing and was analyzed on
an ABIPrism 310 sequencer (Big Dye; PerkinElmer Life Sciences).
Computer-assisted Analysis of Hsc70--
The three-dimensional
models of hsc70 and DnaK were visualized and processed from the PDB
files 1BUP and 1DKG, respectively, using the program RASMOL
(Version 2.6).
Screening of Membrane-bound Peptides with Hap46--
To
investigate the interaction of the molecular chaperones hsp70 and hsc70
with the accessory protein Hap46 we used membrane-bound decapeptides
deduced from the amino acid sequence of the human hsc70 ATP-binding
domain. These peptides were arranged in scans staggered by one amino
acid, beginning at the amino-terminal end and resulting in a total of
370 peptides covering amino acids 1 to 379. For screening we used
recombinant Hap46 and visualized the complexes with monoclonal
antibodies CC9E8 or 3.10G3E2 specific for Hap46/BAG-1 proteins. Both
antibodies yielded very similar interaction patterns. As depicted in
Fig. 1, several distinct peptide areas
of different intensities showed up by
immunostaining. The seven regions of strong interaction signals
are listed in Table I and are shown in blue in Fig.
2, A to G, on the
three-dimensional model of hsc70. Within each of these regions, of
course, interaction with Hap46 may involve only some of the residues
positioned at the surface of the hsp70/hsc70 molecule. The experiments
presented in Fig. 1 were carried out with three independently
synthesized sets of peptides, stained with either antibody, and
resulted in almost identical interaction patterns, whereas control
membranes incubated with antibodies but without Hap46 showed no
reactions above background (data not shown).
Interestingly, peptides covering positions 281 to 289 (Fig. 1,
region VI) lighted up strongly with only one of the
monoclonal antibodies, 3.10G3E2, used in the present study (Fig. 1,
right row). This suggests that the epitope specific for the
other antibody, CC9E8, is not readily accessible once Hap46 is bound to
this peptide sequence. We also need to draw attention to the set of
peptides that constitute region II (cf. Fig. 1
and Table I). They differ from all the others in that they include a
Trp residue and cover the region of hsc70 containing the only Trp
present in the ATP-binding domain. In control experiments we observed
that peptides containing one or several Trp residues reacted very
strongly in our peptide screening assays, almost independent of the
other amino acids and the sequence (data not shown). This coincides
with the experience of others who found that Trp-containing peptides
used in the phage-display approach resulted in exceedingly strong
interactions, easily leading to misinterpretations (35).
Screening of Phage-displayed Random Peptide Libraries with
Hap46--
In an alternative approach we used a peptide library
expressed in fusion with the pIII protein of a derivative of the
filamentous phage M13 (33). This commercially available library had a
complexity of 109 primary transformants presenting random
dodecapeptides. Selection of interacting phage particles was performed
with bacterially expressed Hap46 immobilized on plastic Petri dishes.
Recombinant phage particles retained on this matrix were then amplified
in E. coli and used for further biopanning with Hap46,
carried out in parallel using alternating block and preclearing
conditions to eliminate plastic and block binders. After two and three
rounds of panning, respectively, clones were isolated, and the inserted DNAs were analyzed. As shown in Table II,
the 12-mer peptide sequence QHFNNSVNLGFT was greatly enriched,
representing roughly 70% of the inserts after the second and third
panning rounds. Other sequences, however, were not significantly
enriched.
Upon checking the published amino acid sequences of hsp70 (36) and
hsc70 (37) we did not find the above dodecapeptide QHFNNSVNLGFT
linearly represented in the primary amino acid sequence. We then
searched for homologies within the published three-dimensional structure of hsc70 (9, 11) and indeed detected an almost linear area on
the surface with striking similarities to the respective phage-displayed peptide. Some of the amino acid residues within this
area, however, are quite distantly located in the primary amino acid
sequence. Peptide
QHFNNSVNLGFT is thus
reduced to residues QHFVLGT, corresponding to positions Gln22-His23-Phe21-Val20
and Leu135-Gly136-Thr138 in a
split sequence with opposing polarities in the polypeptide. This in
fact shows that only about half of the amino acid residues of the
dodecapaptide contribute to the molecular interaction on the surface of
the hsc70 molecule. Depicted in red in the model of Fig.
2H, the QHFVLGT area (region A) is located on
subdomain I of the ATP-binding domain of hsc70 and partially overlaps
with region I (amino acids Gln22,
His23, Phe21, and Val20) and
region IV (amino acids Leu135,
Gly136, and Thr138) disclosed by the peptide
scans (Table I).
When we compared the amino acid sequences of hsc70 and hsp70 within the
potential Hap46 interaction sites identified by both approaches we
detected only some minor differences. Conservatively substituted
residues include Val95 Over the past few years, Hap46/BAG-1 proteins have been described
to associate with an enormous variety of completely unrelated mammalian
proteins, like nuclear receptors and other transcription factors (15,
19, 38-41), as well as many other proteins (16, 17, 42-46). However,
the majority of these interactions appear to be mediated by molecular
chaperones of the hsp70 type, which have the ability to promiscuously
associate with all kinds of proteins, in particular if these are
partially misfolded and/or contain hydrophobic areas exposed at the
surface. On the other hand, Hap46 has also been established as a
DNA-binding protein that has the potential to stimulate transcription
(47). The amino-terminal end of Hap46, which contains clusters of
positively charged amino acids, is involved in this activity (47). By
contrast, the carboxyl-terminal portion of Hap46/BAG-1 has been shown
to be required for the interaction with hsp70s (20, 32). However, on
the part of hsp70s, the interaction sites are rather ill-defined. In
Far-Western blots both subdomains I and II of the ATP-binding domain of
human hsp70 reacted with Hap46, but in the yeast two-hybrid system only
one of these subdomains gave a positive result (18). We therefore now
turned to alternative and more sensitive methods with the aim of
obtaining more detailed information about the contact areas within the
ATP-binding domain of hsp70/hsc70.
Membrane-bound peptide libraries designed according to the amino acid
sequence of the ATP-binding domain of human hsc70 were screened with
Hap46 and specific monoclonal antibodies. This resulted in detection of
several potential regions of strong interactions (cf. Table
I). All of these potential contact regions, depicted in blue
in Fig. 2, are located on one side of the ATP-binding domain, except
for region II, which is on the other side of the molecule
(Fig. 2, panel B). We consider the latter region as unlikely to be of any significance for binding of Hap46 because of the fact that
it contains a Trp residue and that tryptophanes were found to trigger
artifactual protein-protein interaction, as described above. In any
event, the peptide-scanning method is able to detect clusters of amino
acids, probably because of the fact that a large number of peptide
molecules is present in a single spot on the membrane in a densely
packed array. Indeed, some of the potential contact regions obtained by
peptide scanning look like patches on the surface of hsc70
(cf. Fig. 2)
In a complementary approach, we used the phage-display technique with a
random dodecapeptide library. In contrast to peptide scanning, phage
M13 presents only a maximum of five peptide copies on the tip of each
particle. Furthermore, detection of interaction sites is restricted by
the limited complexity of the phage library itself, in particular if
rather long polypeptide stretches are inserted, as in our study. In
addition, a multitude of parameters (e.g. target
concentration, phage valency, degree of background binding, and
selection stringency) are known to greatly influence the panning
procedure such that eventually only one binder may dominate the
population of enriched phages (48). On the other hand, the
phage-display technique may uncover rather complex interaction areas
that would never show up as such in the peptide-scanning approach. This
was indeed the case with region A (cf. Fig.
2H). It is a split epitope in that it is not contiguous
within the amino acid sequence of hsp70 or hsc70, and the two
polypeptide segments comprising it are of opposite polarity. On the
surface of hsc70, region A represents an elongated array of
amino acid residues (cf. Fig. 2H).
In the model of Fig. 3, we combined the
results obtained with the membrane-bound peptide-scanning and
phage-display techniques and show the ATP-binding domain of hsc70 in a
front view (A) and turned around by roughly 90°
(B). This presentation clearly shows that all interaction
regions are on one side of the molecule, except for region
II, which remains questionable (see above). The fact that the
other side is void of interaction sites suggests specificity.
Interestingly, region A identified by phage display overlaps
on either end with regions I and IV obtained by
peptide scanning (cf. Fig. 3, A and
B). Regions A, I, and IV,
together with neighboring region III, form a distinct
structure including a prominent protuberance, best seen in Fig.
3B. We think that this area on the surface of hsc70 within
subdomain I is very well suited for protein-protein interactions as it
represents a remarkable structural feature and thus stands out as a
major site of contact with Hap46. A second area of significant
interaction is defined by regions V and VI
located on subdomain II, i.e. on the other side
of the central cleft of the ATP-binding domain. Located in between
these major areas of contact but positioned somewhat to the lower
portion of subdomain II is region VII (Fig. 3A),
which possibly contributes to a much lesser degree to the interaction with Hap46. Nevertheless, it is astonishing at first glance that interaction with Hap46 should involve such large surface areas of
hsp70/hsc70, as outlined in Fig. 3, located on the same side of the
molecule.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, and (iii) 50% ethanol, 10% acetic acid. Upon
incubation with antibodies alone, the very same regions lighted up
(data not shown), demonstrating that Hap46 had not been removed from
the membranes. In independent sets of experiments with
membrane-bound peptides used for epitope mapping of monoclonal
antibodies,1 it was observed
that, when blocking as above, the interacting protein was not removed
from the contact spot if concentrations above 100 ng/ml had been used.
Although the complex Hap46·hsc70 has an equilibrium dissociation
constant in the order of 1 to 100 nM (31, 32) and is
readily formed in the presence of 1 M urea (19), we
expected lower affinities of Hap46 to peptides derived from hsc70. For
the above reasons we only used newly synthesized peptide arrays for our
interaction studies.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (78K):
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Fig. 1.
Membrane-bound peptide scans.
Decapeptides covering amino acids 1 to 383 of the hsc70 ATP-binding
domain were synthesized in arrays as described under "Experimental
Procedures." The very last 4 amino acids (residues 380 to 383) showed
no interaction, and the respective peptides are left out from the
presentation. The left column of peptide spots presents
those stained with antibody CC9E8, and the right column
shows staining with antibody 3.10G3E2. Peptides of strong interaction
with Hap46 are marked in blue, and these regions are labeled
with roman numerals according to their occurrence within the
sequence of hsc70.
Membrane-bound peptides interacting with Hap46
View larger version (42K):
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Fig. 2.
Three-dimensional model of the ATP-binding
domain of hsc70 showing potential Hap46 interacting regions.
Region I (panel A), region II
(panel B), region III (panel C),
region IV (panel D), region V
(panel E), region VI (panel F), and
region VII (panel G), as identified from scans
(cf. Fig. 1), are presented in blue with the
model of the ATP-binding domain of hsc70 turned appropriately.
Region A (panel H), identified by phage display,
is labeled in red. Subdomain I of the ATP-binding domain is
in light blue whereas subdomain II is in
white.
Phage-displayed peptides interacting with Hap46
Ile, Ala98
Gly, Ile282
Leu, Tyr288
Phe, and
Lys356
Arg and are expected not to influence
interactions with Hap46. Less conservative is the Lys137
Tyr exchange in region IV, indicating that these
residues may not significantly contribute to the interaction with
Hap46. Similarly, amino acids Cys17, Val18, and
Gly19 (region I), Val123,
Leu124, Met127, and Ala131
(region III), Ala142 (region IV), and
Cys267 and Leu274 (region V), which
are present in the interacting membrane bound peptides but buried
inside the hsc70 molecule, will probably not participate in the
interaction. However, these residues may stabilize structures required
for interaction.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (38K):
[in a new window]
Fig. 3.
Three-dimensional model of the ATP-binding
domain of hsc70 showing combined results. Potential Hap46
interacting regions identified from membrane-bound peptide scans
(regions I VII; in blue) and by the
phage-display technique (region A) are presented. The
overlap of region A with regions I and
IV is shown in purple. Coloring of hsc70
subdomains is as in Fig. 2. A, front view. B,
model turned by roughly 90°.
The above considerations lead us to compare the potential contact areas
on mammalian hsc70 for the associating protein Hap46 with the
interaction sites on the ATP-binding domain of the bacterial hsp70
counterpart DnaK for its respective regulatory factor GrpE. These were
established by x-ray structural analysis of the complex and showed
several areas of close contact (49) covering a significant surface area
on one side of the molecule. The two largest of them are on the upper
portions of subdomains I and II, respectively, on each side of the
central cleft of the ATP-binding domain. Binding of GrpE was thus shown
to produce a torsion of subdomain II with a relative opening of the
cleft that harbors the ATP-binding site (49). This readily explained
the large stimulation of the ADP/ATP exchange on DnaK upon association
with GrpE (49, 50). The experiments described in the present study
disclosed two major contact areas on hsc70 for the associating factor
Hap46, located on the same side of the ATP-binding domain. One of these
(combined regions A, I, and IV)
corresponds in parts to some of the contact areas on DnaK for binding
of GrpE (49), as depicted in the side by side views of Fig.
4. Most of the amino acid residues
exposed to the respective surfaces are different with the exception of the peptide sequence Tyr-Leu-Gly within region IV, as
highlighted in Fig. 5. The second contact
area on hsc70 (regions V and VI) shows a limited
degree of topological overlap with DnaK on the space-filling model
(Fig. 4) but no similarities within the respective polypeptide
sequences (Fig. 5). As the comparison of Fig. 4 shows, Hap46 and GrpE
touch hsc70 and DnaK, respectively, at somewhat different areas on
subdomain II. These differences are consistent with the fact that GrpE
and Hap46 have no significant homologies in sequence and Hap46 does not
bind to DnaK (19), whereas GrpE is unable to interact with
hsc70,2 just as
antibiotic insect peptides bind to DnaK but not to eukaryotic hsp70
(51). Nevertheless, we expect that Hap46 binding produces a similar
opening of the central cleft in the ATP-binding domain of mammalian
hsp70/hsc70, probably by grasping it on either subdomain, as GrpE does.
In fact, the position of region V (Fig. 4B)
suggests that Hap46 partially reaches into the central cleft for
binding. Recently, an alternative three-dimensional structure of the
hsp70 ATP-binding domain has been identified that shows a distinct
outward shift of the upper part of subdomain II (52). Binding of Hap46 to the hsp70/hsc70 molecular chaperone may then favor this more open
conformation, which, however, can only be proven by a detailed analysis
of crystals of the hsc70·Hap46 complex. Hap46 has been shown to
enhance the nucleotide exchange reaction of hsc70 (21, 24), and in this
respect it resembles GrpE. However, GrpE and Hap46 are certainly not
equivalent regulators of hsp70 chaperones, as the former greatly
stimulates protein refolding reactions involving DnaK in combination
with DnaJ (49, 53) whereas the latter inhibits the respective activity
of hsc70 (18-20, 54).
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Hap46 and Hip/p48 very differently affect the nucleotide binding and
chaperoning activities of hsp70s. Thus Hip/p48 was found to stabilize
the ADP-bound state and to increase the refolding of a denatured model
protein by hsc70 in concert with hsp40 (13) whereas Hap46 increases the
exchange of ATP for ADP prebound to hsc70 and accelerates the
hydrolysis of ATP in the presence of hsp40 (21, 24). Nevertheless,
Hap46 and Hip/p48 compete for binding to the ATP-binding domain (18,
21). Like Hap46, the accessory protein HspBP1 inhibits protein
refolding mediated by hsp70 but, in contrast to Hap46, decreases ATP
binding and inhibits the ATPase activity of hsp70 (22). These
observations suggest that the hsp70-binding proteins Hap46, Hip/p48,
and HspBP1 interact very differently with the ATP-binding domain of
hsp70s. It will thus be interesting to find out more about the contact
sites for Hip/p48 and HspBP1 and to compare them with those described
here for interaction with Hap46.
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ACKNOWLEDGEMENTS |
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We thank Drs. A. Pater (Memorial University of Newfoundland, St. John's, Newfoundland, Canada) and G. Packham (Ludwig Institute for Cancer Research, Imperial College of Medicine, London, United Kingdom) for providing monoclonal antibodies CC9E8 and 3.10G3E2, respectively. We also thank Y. Niyaz and R. Bräuning for help with digital imaging.
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FOOTNOTES |
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* This work was supported by a grant from the Deutsche Forschungsgemeinschaft.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.
§ To whom correspondence should be addressed. Tel.: 49-6221-545256; Fax: 49-6221-545678; E-mail: gabi@sirius.mgen.uni-heidelberg.de.
Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M006967200
1 M. Blüthner and G. Petersen, unpublished results.
2 B. Bukau, personal communication.
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