Mapping of Functional Domains of
-SNAP*
Katsuko
Tani,
Mika
Shibata,
Kazuho
Kawase,
Hoshiko
Kawashima,
Kiyotaka
Hatsuzawa,
Masami
Nagahama, and
Mitsuo
Tagaya
From the School of Life Science, Tokyo University of Pharmacy and
Life Science, Hachioji, Tokyo 192-0392, Japan
Received for publication, December 26, 2002
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ABSTRACT |
-Soluble
N-ethylmaleimide-sensitive factor (NSF) attachment protein
(
-SNAP) is capable of stabilizing a 20 S complex consisting of NSF,
-SNAP, and SNAP receptors (SNAREs), but its function in vesicular
transport is not fully understood. Our two-hybrid analysis revealed
that
-SNAP, unlike
-SNAP, interacts directly with NSF, as well as
Gaf-1/Rip11, but not with SNAREs. Gaf-1/Rip11 is a
-SNAP-associated
factor that belongs to the Rab11-interacting protein family. To gain
insight into the molecular basis for the interactions of
-SNAP with
NSF and Gaf-1/Rip11, we determined the regions of the three proteins
involved in protein-protein interactions.
-SNAP bound to NSF via its
extreme C-terminal region, and the full-length NSF was needed to
interact with
-SNAP. Both the N-terminal and C-terminal regions of
-SNAP were required for the binding to Gaf-1/Rip11. Gaf-1/Rip11
bound to
-SNAP via its C-terminal domain comprising a putative
coiled-coil region. Although the C-terminal domain of Gaf-1/Rip11 also
interacts with Rab11, the binding of
-SNAP and Rab11 to Gaf-1/Rip11
was not mutually exclusive. Rather, Gaf-1/Rip11 was capable of serving a link between
-SNAP and Rab11. A complex comprising
-SNAP and Gaf-1/Rip11 was disassembled in a process coupled to NSF-mediated ATP
hydrolysis, suggesting that the interaction between
-SNAP and
Gaf-1/Rip11 is of functional significance.
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INTRODUCTION |
Many membrane fusion events occurring in membrane trafficking in
eukaryotic cells require a common set of proteins such as N-ethylmaleimide-sensitive factor
(NSF)1 and SNAPs and membrane
compartment-specific proteins consisting of complementary sets of
coiled-coil proteins called SNAREs (1-4). Although the precise
molecular mechanism for membrane fusion is not fully understood, one
current model predicts that membrane fusion is driven by a tight
binding between vesicle-associated SNAREs (the VAMP/synaptobrevin
family) and target membrane-associated SNAREs (the syntaxin and SNAP-25
families) (5). SNAREs per se encode compartmental
specificity of membrane fusion (6). Following membrane fusion, the
cis (same membrane) SNARE complex is disassembled by the
chaperone ATPase NSF (2). SNAPs not only serve as a linkage between NSF
and the cis SNARE complex (7, 8) but also couple the energy
produced by NSF-mediated ATP hydrolysis to a conformational change in
syntaxin of the cis SNARE complex (9), which leads to the
disassembly of the NSF-SNAPs-SNAREs complex (7, 8).
The SNAP family consists of ubiquitously expressed
and
isoforms
and a brain-specific
isoform (10). So far
-SNAP has been studied
most extensively.
-SNAP stimulates the ATPase activity of NSF (11)
by interacting via its N-terminal and extreme C-terminal regions (12)
and is essential for a wide variety of membrane fusion events including
intra-Golgi protein transport (13). Based on the results of the x-ray
crystallographic analysis of Sec17p, the yeast ortholog of
-SNAP
(13, 14), it has been proposed that
-SNAP (Sec17p) functions as
lever arms that transmit forces generated by a conformational change in
NSF to SNAREs (15). Given the high sequence similarity between
-SNAP
and
-SNAP, it is postulated that the function of
-SNAP is quite
similar to that of
-SNAP. On the other hand, the physiological
significance of
-SNAP is much less documented. Although
-SNAP
enhances the secretion of neurotransmitters from several types of cells
(16, 17), it exhibits a very low transport activity in an intra-Golgi protein transport assay (13) and is not required for transport from the
endoplasmic reticulum to the Golgi (18).
-SNAP stabilizes the 20 S
NSF-
-SNAP-SNAREs complex but cannot substitute for
-SNAP in
complex formation (19).
Chen et al. (20) reported that Gaf-1
(
-SNAP-associated
factor-1) encoded by the clone KIAA0857 (21)
interacts with
-SNAP and is localized in the mitochondria.
Independently, Prekeris et al. (22) demonstrated that the
same protein but differently named Rip11
(Rab11-interacting protein),
because of its ability to bind to Rab11, is localized in recycling
endosomes and regulates apical membrane trafficking by interacting with
Rab11 in polarized epithelial cells. Recent studies revealed that
Gaf-1/Rip11 and several other proteins constitute a family of
Rab11-interacting proteins (22-27).
In the present study we extensively characterized the interactions of
-SNAP with NSF and Gaf-1/Rip11. The results suggest that
-SNAP
interacts with NSF via its extreme C-terminal region, whereas the
N-terminal and C-terminal regions are required for the interaction with
Gaf-1/Rip11. In addition, we showed a complex comprising
-SNAP and
Gaf-1/Rip11 is disassembled in a process coupled to NSF-mediated ATP hydrolysis.
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EXPERIMENTAL PROCEDURES |
Antibodies--
A Gaf-1/Rip11 fragment (amino acids 524-653)
and the full-length
-SNAP were expressed as His6-tagged
proteins in Escherichia coli cells and purified
using a nickel-nitrilotriacetic acid-agarose column (Qiagen). The
isolated proteins were injected into rabbits to produce antisera.
Monoclonal antibodies against Rab11 (clone 47; Transduction
Laboratories),
-tubulin (GTU-88; Accurate Chemical and Scientific
Co.), FLAG (M2; Sigma), and GST (Clontech) were obtained. A polyclonal antibody against GST was obtained from Santa
Cruz Biotechnologies. A monoclonal antibody against c-Myc (clone
9E10) was purified from ascitic fluid and cross-linked to protein G
beads using dimethyl pimelimidate. A monoclonal antibody against NSF
(2E5) was prepared as described (28).
Plasmids--
pQE9, pQE30, and pQE31 were purchased from Qiagen
and used for the expression of His6-tagged proteins.
pGEX-4T-1 was obtained from Amersham Biosciences and used for
the expression of GST fusion proteins. The clone KIAA0857 was obtained
from the Kazusa DNA Research Institute, Kisarazu, Japan. pEBG
(29) and pFLAG-CMV2 (Eastman Kodak Co.) were used for the expression of
proteins in mammalian cells. The cDNA encoding
-SNAP was a
generous gift from Dr. S. W. Whiteheart (University of
Kentucky College of Medicine). The plasmids for Myc-tagged Rab11Q70L
and Rab11S25N were kindly donated by Dr. Y. Takai (Osaka University).
Two-hybrid Analysis--
The full-length cDNA of
-SNAP
was inserted into pGBT9, and the resultant plasmid was used as bait. A
GAL4 DNA activation domain fusion library in pACT2 (MATCHMAKER human
kidney cDNA library; Clontech) was used to
isolate interacting clones. Yeast colonies that grew in medium lacking
histidine were picked up, and their
-galactosidase activity was
assayed on filters. To examine protein-protein interactions, cDNAs
for
-SNAP and
-SNAP were inserted into the bait plasmid pGBT9,
and cDNAs for NSF and Gaf-1/Rip11 were inserted into the prey
plasmid pGAD424.
-Galactosidase activity was measured after
transformation of yeast with the indicated combinations of the bait and
prey plasmids.
Pull-down Experiments Using Recombinant
Proteins--
Recombinant GST proteins were mixed with 10 µl of
glutathione-Sepharose 4B and then the mixture was gently rotated at
4 °C for 30 min. After the beads were washed, they were incubated
with His6-tagged proteins in incubation buffer comprising
20 mM HEPES/KOH, pH 7.0, 50 mM KCl, 2 mM mercaptoethanol, 0.5 mM ATP, 10% glycerol, and 1 mg/ml bovine serum albumin at 4 °C for 1 h with gentle
rotation. The beads were washed three times with incubation buffer
devoid of bovine serum albumin, and the bound proteins were eluted with 50 mM Tris-HCl, pH 8.0, with 5 mM glutathione.
The eluted proteins were concentrated by trichloroacetic
acid-deoxycholate precipitation and then subjected to SDS-PAGE on 10%
gels. Gels were stained with Coomassie Brilliant Blue R-250.
Immunoprecipitation and Pull-down Experiments in a Mammalian
Expression System--
Transfection of cells and preparation of cell
lysates were performed as described (30). For co-transfection, plasmids
(1 µg each) were used. At 20 h after transfection, cells plated
on 35-mm dishes were washed and then lysed in 300 µl of lysis buffer comprising 25 mM HEPES/KOH, pH 7.2, 1% Triton X-100, 150 mM KCl, 0.5 µg/ml leupeptin, 2 µM
pepstatin, 2 µg/ml aprotinin, 2 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol. The lysates were clarified by centrifugation. To the supernatants was added
3-5 µg of an antibody. After 30 min on ice, 10 µl of protein
G-Sepharose 4B was added to the mixtures and then incubated at 4 °C
for 2 h with gentle rotation. The beads were washed with lysis
buffer several times and then the bound proteins were eluted by boiling
in 10 µl of 2× SDS-PAGE sample buffer. For pull-down experiments,
cell lysates were mixed with 10 µl of glutathione-Sepharose 4B and
then incubated at 4 °C for 2 h with gentle rotation. The beads
were washed, and the bound proteins were eluted as described above.
Immunoblotting--
Proteins were separated by SDS-PAGE and then
transferred to nitrocellulose membranes. After incubation with
appropriate primary antibodies and then with horseradish
peroxidase-conjugated secondary antibodies, the membranes were
developed on autoradiographic film by enhanced chemiluminescence
(Amersham Biosciences).
Preparation of Rat Liver Membranes--
Livers were removed from
starved rats, rinsed, perfused, and weighed. They were cut into small
pieces and homogenized with a Potter-Elvehjem homogenizer (5 strokes)
in 4 volumes of homogenization buffer comprising 25 mM
HEPES/KOH, pH 7.2, 100 mM KCl, 0.5 µg/ml leupeptin, 2 µM pepstatin, 2 µg/ml aprotinin, 2 mM EDTA,
1 mM phenylmethylsulfonyl fluoride, and 1 mM
dithiothreitol. The homogenate was centrifuged at 3,000 rpm (Tomy TA-4
rotor) for 10 min and then at 10,000 rpm for 10 min. The supernatant
was centrifuged at 33,000 rpm (Beckman 50.2Ti rotor) for 1 h to
recover membranes.
Velocity Sedimentation--
Velocity sedimentation was performed
essentially as described (7) with a slight modification. Rat liver
membranes (1.0 mg) were solubilized with 1% Triton X-100, and
insoluble materials were removed by centrifugation. The membrane
extracts (220 µl) were mixed with His6-tagged NSF (3 µg) and His6-tagged
-SNAP (0.7 µg), and the mixture
was incubated on ice for 30 min. When indicated, 1 M
MgCl2 was added to the mixture at a final concentration of
8 mM. The mixture was layered on the top of glycerol
gradients consisting of 0.4 ml each of gradient buffer containing 35, 32.5, 30, 27.5, 25, 22.5, 20, 17.5, 15, and 12.5% glycerol (w/v).
After centrifugation at 34,000 rpm (Beckman SW50 rotor) for 13 h,
fractions (0.4 ml each) were recovered from the top. The proteins of
the gradient fractions were precipitated with trichloroacetic acid and
deoxycholate, washed with acetone, and then dissolved in SDS-PAGE sample buffer.
Disassembly of a Complex Containing
GST-Gaf-1/Rip11 and
-SNAP--
Cells expressing
GST-Gaf-1/Rip11 and FLAG-tagged
-SNAP, cultured on 35-mm dishes,
were lysed in 300 µl of lysis buffer. The lysates were mixed with an
equal volume of cell lysates separately prepared from non-transfected
cells. When indicated, 1 M MgCl2 was added to
the mixture at a final concentration of 8 mM. The mixture
was incubated at 16 °C for 1 h and then GST-Gaf-1/Rip11 was
pulled down with glutathione beads. The proteins pulled down were
subjected to SDS-PAGE and analyzed by immunoblotting with antibodies
against GST and FLAG.
For the preparation of NEM-treated cell lysates, non-transfected cells
were lysed in lysis buffer devoid of dithiothreitol. The cell lysates
were incubated with 1 mM NEM on ice for 15 min followed by
15 min treatment with 2 mM dithiothreitol to quench excess
NEM.
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RESULTS |
Screen of
-SNAP-interacting Proteins--
To identify
-SNAP-interacting proteins other than Gaf-1/Rip11 (20), we screened
a human kidney expression library using the full-length
-SNAP as
bait. Screening two times yielded 68 positive clones. Among the clones
obtained, 20 clones encoded Gaf-1/Rip11, and 24 appeared to encode its
alternative splice variant, because the encoded protein possesses the
same C-terminal 131 amino acid residues as Gaf-1/Rip11 but has a
different N-terminal sequence. Characterization of the putative splice
variant will be described elsewhere. Fifteen clones were found to
comprise the entire coding region of NSF. Proteins encoded by the other nine clones were different from one another. One was cytosolic thiolase, which was also identified as a potential
-SNAP-interacting protein in a two-hybrid screen by Chen et al. (20).
Interaction between
-SNAP and NSF--
Because the direct
interaction between
-SNAP and NSF was not reported previously, we
decided to analyze the interaction in detail. We first generated
-SNAP mutants encoding the N-terminal half (amino acids 1-152) and
the C-terminal one (amino acids 153-312). A two-hybrid assay revealed
that the C-terminal region is responsible for the interaction with NSF
(Fig. 1A). We then generated a
series of mutants with C-terminal deletions. When two amino acids were removed from the C terminus, the interaction between
-SNAP and NSF
was abolished. To examine whether the two C-terminal residues, Leu-311
and Cys-312, directly contribute to the interaction with NSF, we
replaced them individually and both by Ala via site-directed mutagenesis. The results showed that both the residues are important for the interaction with NSF (Fig. 1A).

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Fig. 1.
Mapping of the binding site for NSF
on -SNAP. A, two-hybrid
analysis. Full-length or mutant -SNAP cDNA fragments and
full-length NSF cDNA were inserted into pGBT9 and pGAD424,
respectively. The interaction of -SNAP or its mutants with NSF in
yeast was examined using a -galactosidase filter assay.
B, pull-down experiments. Coomassie-stained gel of isolated
His6-tagged NSF (lane 1), GST (lane
2), GST- -SNAP (full-length; -SNAP(F)) (lane
3), GST- -SNAP (amino acids 1-152; -SNAP(N))
(lane 4), and GST- -SNAP (amino acids 153-312;
-SNAP(C)) (lane 5). To analyze protein-protein
interactions, His6-tagged full-length NSF (10 µg) was
mixed with glutathione-Sepharose 4B that had been incubated with 12 µg of GST (lane 6), GST- -SNAP (full-length) (lane
7), GST- -SNAP (amino acids 1-152) (lane 8), or
GST- -SNAP (amino acids 153-312) (lane 9). The bound
proteins were eluted with glutathione, separated by SDS-PAGE, and
stained. C, pull-down experiments. Coomassie-stained gel of
isolated NSF (lane 1), GST (lane 2), GST- -SNAP
(wild-type) (lane 3), GST- -SNAP mutant in which Leu-311
was replaced by Ala (L311A; lane 4), and GST- -SNAP mutant
in which Cys-312 was replaced by Ala (C312A; lane 5). To
analyze protein-protein interactions, His6-tagged
full-length NSF (10 µg) was mixed with glutathione-Sepharose 4B that
had been incubated with 12 µg of GST (lane 6),
GST- -SNAP (wild-type) (lane 7), GST- -SNAP (L311A)
(lane 8), or GST- -SNAP (C312A) (lane 9). The
bound proteins were eluted with glutathione, separated by SDS-PAGE, and
stained.
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To confirm the results of the two-hybrid analysis, we performed an
in vitro binding assay using isolated bacterial proteins. To
facilitate purification, NSF was expressed as a His6-tagged protein, and
-SNAP was expressed as a GST fusion protein. Consistent with the results of the two-hybrid analysis, His6-tagged
NSF was pulled down with GST-
-SNAP (full-length) (Fig.
1B, lane 7) and GST-
-SNAP (amino acids
153-312) (lane 9) but not with GST-
-SNAP (amino acids
1-152) (lane 8). In addition, His6-tagged NSF
was not pulled down with GST-
-SNAP in which Leu-311 was replaced by
Ala (L311A) (Fig. 1C, lane 8). Although
His6-tagged NSF was pulled down with GST-
-SNAP in which
Cys-312 was replaced by Ala (C312A) (lane 9), the amount was
smaller than that pulled down with wild-type GST-
-SNAP. These
results clearly showed that the extreme C-terminal region, especially
Leu-311, is important for the interaction with NSF.
We next determined the binding site for
-SNAP on NSF. NSF can be
divided into three domains, N-terminal and two ATP-binding domains
referred to as D1 and D2 (28, 31, 32). Two-hybrid analysis and in
vitro binding experiments revealed that the full-length NSF, but
neither the N-terminal domain (amino acids 1-205) nor the D1D2 domain
(amino acids 206-744), binds to
-SNAP (Fig.
2). These results are consistent with the
fact that only full-length NSF clones were obtained in the two-hybrid
screen.

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Fig. 2.
Full-length NSF is required for the
interaction with -SNAP. A,
two-hybrid analysis. Full-length -SNAP cDNA and full-length or
truncated NSF cDNA fragments were inserted into pGBT9 and pGAD424,
respectively. The interaction of -SNAP with NSF or its fragments in
yeast was examined using a -galactosidase filter assay.
B, pull-down experiments. Coomassie-stained gel of isolated
His6-tagged NSF (lane 1), the N-domain (amino
acids 1-205; lane 2), the D1D2 domain (amino acids
206-744; lane 3), GST (lane 4), and GST- -SNAP
(lane 5). To analyze protein-protein interactions, 10 µg
of His6-tagged full-length NSF (lanes 6 and
7), the N-domain (lane 8), or the D1D2-domain
(lane 9) was mixed with glutathione-Sepharose 4B that had
been incubated with 12 µg of GST (lane 6) or GST- -SNAP
(full-length) (lanes 7, 8, and 9). The
bound proteins were eluted with glutathione, separated by SDS-PAGE, and
stained.
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-SNAP Does Not Bind to SNAREs--
-SNAP interacts primarily
with syntaxin and weakly with SNAP-25 (9, 33). Our previous results
(34) demonstrated that SNAREs can interact with
-SNAP in the yeast
two-hybrid system even though they possess membrane anchors. The fact
that clones encoding SNAREs were not obtained in the two-hybrid screen
using
-SNAP as bait may suggest that
-SNAP does not interact
directly with SNAREs. Indeed, a two-hybrid assay demonstrated that
-SNAP does not interact with various syntaxins, cellubrevin (a
ubiquitously expressed protein of the VAMP-2 family) (35), or SNAP-23
(a ubiquitously expressed protein of the SNAP-25 family) (36)
(Table I). These observations raised the
possibility that
-SNAP is incorporated into the NSF-
-SNAP-SNAREs
complex through binding to NSF. To test this possibility, we carried
out velocity sedimentation analysis. Triton X-100 extracts of rat liver
membranes were incubated with purified recombinant His6-NSF
and His6-
-SNAP in the presence or absence of
Mg2+-ATP, and the mixtures were then subjected to velocity
centrifugation. As shown in Fig.
3A, significant amounts of
endogenous and recombinant His6-
-SNAP co-sedimented with
NSF in the absence of Mg2+ (fractions 7-9). In contrast,
no co-sedimentation was observed in the presence of Mg2+
(Fig. 3B). These results were essentially the same as those
reported previously (7). In the case of a
-SNAP mutant (L311A) that is defective in the ability to bind to NSF, no co-sedimentation with
NSF was observed even in the absence of Mg2+ (Fig.
3C), suggesting that the interaction with NSF is essential for
-SNAP to be incorporated into the 20 S complex.
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Table I
Interaction between SNAPs and SNAREs in the two-hybrid system
-SNAP and -SNAP were inserted into pGBT9 (bait plasmid), and
SNAREs were inserted into pGAD424 (prey plasmid). After
co-transformation of yeast with the indicated combinations of the bait
and prey plasmids, -galactosidase activity was measured.
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Fig. 3.
-SNAP is incorporated into a 20 S complex through binding to NSF. Triton X-100 extracts of rat
liver membranes were incubated with His6-tagged NSF and
His6-tagged -SNAP in the absence (A) or
presence (B) of MgCl2 and then subjected to
velocity sedimentation analysis. Alternatively, the extracts were
incubated with His6-tagged NSF and a
His6-tagged -SNAP mutant (L311A) in the absence of
MgCl2 (C). After centrifugation, the proteins in
each fraction were precipitated, separated by SDS-PAGE, and analyzed by
immunoblotting with anti-NSF and anti- -SNAP antibodies. NSF in
fractions 2-4 may represent partially unfolded or degraded ones. NSF
sedimented more slowly in the presence of Mg2+
(B) than its absence (A), reflecting the
disassembly of the NSF-SNAPs-SNAREs complex in the presence of
Mg2+. Two major bands recognized by the anti- -SNAP
antibody correspond to His6-tagged -SNAP (slower
migrating band on gels) and endogenous -SNAP (faster migrating band
on gels), respectively. Note that the -SNAP mutant did not
co-sediment with NSF in the absence of Mg2+, whereas the
endogenous one did (C).
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Interaction between
-SNAP and Gaf-1/Rip11--
To
gain insight into the mechanism governing the interaction between
-SNAP and Gaf-1/Rip11, we determined the region of Gaf-1/Rip11 involved in the association with
-SNAP. The binding site for
-SNAP on Gaf-1/Rip11 must be within its C-terminal 131 amino acids,
because the C-terminal region common to Gaf-1/Rip11 and its putative
alternative splice variant interacts with
-SNAP in the two-hybrid
system. We constructed a series of C-terminal fragments of Gaf-1/Rip11
and performed a yeast two-hybrid assay using the full-length
-SNAP
as bait (Fig. 4A). The results
confined the region responsible for the interaction with
-SNAP to
amino acids 590-640 of Gaf-1/Rip11. This region comprises a putative coiled-coil region (amino acids 608-640), but the coiled-coil region
alone was not sufficient to interact with
-SNAP. To confirm this
observation, we constructed plasmids to express amino acids 590-640
and 608-640 of Gaf-1/Rip11 as GST fusion proteins and
-SNAP as a
FLAG-tagged protein and performed pull-down experiments after
co-transfection of the plasmids into 293T cells (Fig. 4B). Consistent with the results of the yeast two-hybrid analysis, FLAG-tagged
-SNAP was pulled down with the GST fusion protein comprising amino acids 590-640 of Gaf-1/Rip11 (lane 6) but
not with that comprising amino acids 608-640 (lane 5).

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Fig. 4.
The C-terminal region common to Gaf-1/Rip11
and its alternative splice variant is involved in the interaction
with -SNAP. A, two-hybrid
analysis. Full-length -SNAP cDNA and full-length or truncated
Gaf-1/Rip11 cDNA fragments were inserted into pGBT9 and pAD424,
respectively. The interaction between -SNAP and Gaf-1/Rip11 or
truncated ones in yeast was examined using a -galactosidase filter
assay. C2 represents the C2-domain. Hatched and
filled boxes denote the C-terminal region common to
Gaf-1/Rip11 and its alternative splice variant and the putative
coiled-coil region, respectively. B, interaction between
-SNAP and truncated Gaf-1/Rip11 constructs in mammalian cells. 293T
cells were transfected with a plasmid encoding GST (lanes 1 and 4) or a Gaf-1/Rip11 fragment corresponding to amino
acids 608-640 (lanes 2 and 5) or 590-640
(lanes 3 and 6) fused to GST, in addition to a
plasmid for FLAG-tagged -SNAP. The GST fusion proteins in cell
lysates were pulled down, and the precipitated proteins were analyzed
by immunoblotting with antibodies against -tubulin, FLAG, and GST
(lanes 4-6). Five percent input was also analyzed
(lanes 1-3).
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A previous study revealed that Gaf-1/Rip11 is associated with
-tubulin (20). Interestingly, endogenous
-tubulin was efficiently pulled down with the construct comprising amino acids 608-640, which
could not interact with
-SNAP, suggesting that this region is
sufficient to interact with
-tubulin. The fact that the amount of
-tubulin bound to the construct containing amino acids 590-640 of
Gaf-1/Rip11 was trivial compared with that bound to the construct containing amino acids 608-640 may imply that expressed FLAG-tagged
-SNAP competed with endogenous
-tubulin for the binding to the construct containing amino acids 590-640 of Gaf-1/Rip11.
To determine the region of
-SNAP involved in the interaction with
Gaf-1/Rip11, we performed a two-hybrid assay using
-SNAP or its
deletion mutants as bait. The results are summarized in Fig.
5A. Deletion of N-terminal 23 amino acids or C-terminal 89 amino acids eliminated the interaction
with Gaf-1/Rip11. Similar results were obtained when pull-down
experiments were conducted using a mammalian expression system (Fig.
5B).

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Fig. 5.
Mapping of the Gaf-1/Rip11-binding sites
on -SNAP. A, two-hybrid
analysis. Full-length or truncated -SNAP cDNA fragments and
full-length Gaf-1/Rip11 were inserted into pGBT9 and pGAD424,
respectively. The interaction of -SNAP or truncated ones with
Gaf-1/Rip11 in yeast was examined using a -galactosidase filter
assay. Although two -SNAP fragments (amino acids 1-245 and 1-268)
exhibited weak positive signals in the absence of the prey vector,
their signals were considerably stronger in the presence of the prey
vector. B, interaction of -SNAP or its mutants with
Gaf-1/Rip11 in mammalian cells. 293T cells were co-transfected with
plasmids for GST-Gaf-1/Rip11 and FLAG-tagged full-length -SNAP
(lanes 1 and 8) or fragments corresponding to
amino acids 10-312 (lanes 2 and 9), 24-312
(lanes 3 and 10), 1-223 (lanes 4 and
11), 1-245 (lanes 5 and 12), 1-268
(lane 6 and 13), or 1-295 (lanes 7 and 14). GST-Gaf-1/Rip11 in cell lysates was pulled down,
and the precipitated proteins were analyzed by immunoblotting with
antibodies against GST and FLAG (lanes 8-14). Two percent
input was also analyzed (lanes 1-7).
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Gaf-1/Rip11 Is Able to Link
-SNAP to
Rab11--
Members of the Rab11-interacting protein family share a
highly conserved region of 20 amino acids at the C terminus (22-26). Prekeris et al. (23) showed that a peptide corresponding to amino acids 628-645 of Gaf-1/Rip11 competes with Gaf-1/Rip11 protein for the binding to Rab11. Because the Rab11-binding domain on Gaf-1/Rip11 markedly overlaps with the
-SNAP-binding domain, we
wondered whether the
-SNAP-binding domain can interact with Rab11.
Myc-tagged Rab11 proteins and the full-length Gaf-1/Rip11 or its
fragments fused to GST were expressed in 293T cells, and immunoprecipitation was performed by using an anti-Myc antibody. As
shown in Fig. 6A, the
-SNAP-binding domain (amino acids 590-640 of Gaf-1/Rip11) can
interact with the constitutively active Rab11, Rab11Q70L (lane
9), although its interaction was markedly weaker than the
interaction of the full-length Gaf-1/Rip11 (lane 10). Perhaps the C-terminal sequence (amino acids 641-645) of the
Rab11-binding domain significantly contributes to the binding to Rab11.
No interaction was observed with the inactive Rab11, Rab11S25N
(lane 12). The coiled-coil region responsible for the
interaction with
-tubulin (amino acids 608-640 of Gaf-1/Rip11) did
not bind to Rab11Q70L (lane 8).

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Fig. 6.
Interaction between Gaf-1/Rip11 and
Rab11. A, the C-terminal region of Gaf-1/Rip11 is
involved in the interaction with Rab11. 293T cells were transfected
with a plasmid encoding GST (lane 1), Gaf-1/Rip11 fragment
corresponding to amino acids 608-640 (lanes 2 and
5), 590-640 (lanes 3 and 6), or
full-length Gaf-1/Rip11 (lane 4) fused to GST, in addition
to a plasmid encoding Rab11Q70L (lanes 1-4) or Rab11S25N
(lanes 5 and 6) tagged with Myc. The expression
levels of Myc-tagged Rab proteins were comparable (data not shown).
Myc-tagged Rab proteins were immunoprecipitated, and the precipitated
proteins were analyzed by immunoblotting with an anti-GST antibody
(lanes 7-12). Five percent input was also analyzed
(lanes 1-6). HC and LC denote
immunoglobulin heavy chain and light chain, respectively. B,
Gaf-1/Rip11 can link Rab11 to -SNAP. 293T cells were co-transfected
with plasmids for Gaf-1/Rip11 (lanes 1, 2,
5, and 6), Myc-tagged Rab11Q70L (lanes
1, 3, 5, and 7) or Rab11S25N
(lanes 2, 4, 6, and 8), and
FLAG-tagged -SNAP (lanes 1-8). Myc-tagged Rab proteins
were immunoprecipitated with an anti-Myc antibody cross-linked to
protein G beads, and the precipitated proteins were analyzed by
immunoblotting with antibodies against Gaf-1/Rip11, FLAG, and Myc
(lanes 5-8). Five percent input was also analyzed
(lanes 1-4).
|
|
Given that Rab11 and
-SNAP occupy the same region on Gaf-1/Rip11, we
predicted that
-SNAP inhibits the binding of Rab11 to Gaf-1/Rip11 in
a competitive manner. However, when Myc-tagged Rab11Q70L, Gaf-1/Rip11,
and FLAG-tagged
-SNAP were co-expressed, Gaf-1/Rip11 was still
co-precipitated with an anti-Myc antibody (Fig. 6B,
lane 5). Surprisingly, a small but significant amount of
FLAG-tagged
-SNAP was also co-precipitated with Myc-Rab11Q70L. On
the other hand, no co-precipitation of FLAG-tagged
-SNAP was observed with Myc-Rab11S25N even in the presence of Gaf-1/Rip11 (lane 6). It should be noted that expression of Gaf-1/Rip11
is prerequisite for the co-precipitation of
-SNAP with Rab11Q70L (lane 7). These results suggest that Gaf-1/Rip11 is able to
link between Rab11 and
-SNAP, although the linkage efficiency is not high.
A Complex Containing
-SNAP and Gaf-1/Rip11 Is
Disassembled in a Mg2+-ATP-, NSF-dependent
Manner--
The 20 S NSF-SNAPs-SNAREs complex is disassembled in a
process coupled to NSF-mediated ATP hydrolysis (7). This most likely reflects disassembly of the cis SNARE complex after membrane
fusion (1-4). We were interested in whether the binding between
-SNAP and Gaf-1/Rip11 is also affected by NSF and
-SNAP in a
Mg2+-ATP-dependent manner. To address this
question, Triton X-100 extracts prepared from cells expressing
FLAG-tagged
-SNAP and GST-Gaf-1/Rip11 were mixed with lysates
separately prepared from non-transfected cells, and the mixtures were
incubated in the presence of EDTA-ATP or Mg2+-ATP.
GST-Gaf-1/Rip11 in the mixtures was pulled down, and the precipitated
proteins were analyzed by immunoblotting. As shown in Fig.
7, addition of Mg2+-ATP
caused the dissociation of FLAG-tagged
-SNAP from GST-Gaf-1/Rip11 (lane 9), implying the disassembly of the
-SNAP-Gaf-1/Rip11 complex. Addition of cell lysates from
non-transfected cells was necessary for efficient disassembly. It
should be noted that FLAG-tagged
-SNAP and GST-Gaf-1/Rip11 were
overexpressed. Perhaps the cell lysates may supply membrane and
cytosolic proteins required for the disassembly reaction under
conditions in which
-SNAP and Gaf-1/Rip11 were present at high
levels. When cell lysates were pretreated with 1 mM NEM, no
dissociation of FLAG-tagged
-SNAP from GST-Gaf-1/Rip11 occurred
(lane 13), indicating the requirement of NEM-sensitive
component(s) for this process. Addition of His6-tagged NSF
and His6-tagged
-SNAP restored the dissociation reaction (lane 15). These results unequivocally showed the
requirement of NSF for the disassembly of the
-SNAP-Gaf-1/Rip11
complex.

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|
Fig. 7.
NSF-mediated disassembly of the
-SNAP-Gaf-1/Rip11 complex. Lysates from cells
expressing FLAG-tagged -SNAP and GST-Gaf-1/Rip11 were mixed with
non-transfected cell lysates (lanes 1-4 and
9-12) or NEM-treated lysates of non-transfected cells
(lanes 5-8 and 13-16). The mixture was
incubated in the presence of EDTA-ATP (lanes 2,
4, 6, 8, 10, 12,
14, and 16) or Mg2+-ATP (lanes
1, 3, 5, 7, 9,
11, 13, and 15) without (lanes
1, 2, 5, 6, 9,
10, 13, and 14) or with 0.5 µg of
His6-tagged NSF and 0.5 µg of His6-tagged
-SNAP (lanes 3, 4, 7, 8,
11, 12, 15, and 16).
GST-Gaf-1/Rip11 was pulled down, and the precipitated proteins were
analyzed by immunoblotting with antibodies against GST and FLAG
(lanes 9-16). Two percent input was also analyzed
(lanes 1-8).
|
|
 |
DISCUSSION |
In the present study we analyzed the interactions of
-SNAP with
NSF and Gaf-1/Rip11 in detail using the yeast two-hybrid system and
isolated recombinant proteins or a mammalian expression system. Our
results demonstrated that the extreme C-terminal region of
-SNAP,
especially Leu-311, is required for the interaction with NSF, and the
N-terminal 23 amino acids and C-terminal 89 amino acids of
-SNAP
contribute to the binding to Gaf-1/Rip11. For the interaction with
-SNAP, on the other hand, the entire coding region of NSF and the
C-terminal region (amino acids 590-640) of Gaf-1/Rip11 are required.
Although
-SNAP mediates the attachment of NSF to SNAREs (7, 8), it
appears that
-SNAP interacts with NSF only when it binds to SNAREs
or plastic (12, 13, 37). Binding of
-SNAP to plastic may cause a
conformational change in
-SNAP similar to that occurring upon
binding to SNAREs. Consistent with the idea that SNAREs are required
for the interaction of
-SNAP with NSF, no clones encoding NSF were
obtained in our previous two-hybrid screen using
-SNAP as bait (34).
Indeed, our yeast two-hybrid assay showed that
-SNAP hardly
interacts with NSF (data not shown). In contrast,
-SNAP interacts
with NSF in the absence of SNAREs. Despite this difference in the
interactions between the two species of SNAPs and NSF, the penultimate
leucine residues of the SNAPs (Leu-294 in
-SNAP and Leu-311 in
-SNAP) are important for the binding to NSF. In comparison with
-SNAP,
-SNAP appears to have a 22-amino acid insertion between
the putative C-terminal
-helix, which corresponds to loop
14 in
Sec17p (15), and the penultimate leucine. This extra sequence may shift
the position of Leu-311 such that it can interact with NSF in the
absence of SNAREs. Interestingly, replacements of the penultimate
leucine residues cause opposite effects in terms of the binding to NSF.
An
-SNAP mutant in which Leu-294 is replaced by Ala is able to
interact with NSF in a 20 S complex but unable to fully disassemble the
complex in the presence of Mg2+-ATP (12). In contrast, the
corresponding
-SNAP mutant is unable to bind to NSF.
Gaf-1/Rip11 is a member of the recently identified Rab11-interacting
protein family (22-27). The highly conserved C-terminal region of
Rab11-interacting protein family members is responsible for the binding
to Rab11. In the present study we revealed that the same or markedly
overlapping region is capable of interacting with
-SNAP. Despite the
fact that Rab11 and
-SNAP bind to the overlapping site on
Gaf-1/Rip11, overexpression of
-SNAP did not inhibit the binding of
expressed Rab11 to Gaf-1/Rip11. Rather, a significant amount of
-SNAP was co-precipitated with Rab11 in the presence of Gaf-1/Rip11,
suggesting that Gaf-1/Rip11 is able to serve as a link between
-SNAP
and Rab11. How can Gaf-1/Rip11 connect
-SNAP to Rab11? Members of
the Rab11-interacting protein family including Gaf-1/Rip11 are known to
possess extensive homo- and hetero-interacting abilities (27). Using
these abilities Gaf-1/Rip11 may constitute a scaffold or a hydrophobic
patch (38) that can accommodate Rab11 and
-SNAP. Obviously, members
of the Rab11-interacting protein family bind many proteins. Rab11-FIP2, a member of the family, interacts not only with Rab11 but also with
proteins involved in recycling and endocytosis such as myosin Vb,
Reps1, and
-adaptin (24, 39). A previous study (20), as well as the
present one, showed that Gaf-1/Rip11 binds
-tubulin.
Both N-terminal and C-terminal regions of
-SNAP contribute to the
binding to Gaf-1/Rip11. Interestingly, the corresponding regions of
-SNAP are known to interact with SNAREs (9, 40). Because
-SNAP
does not bind SNAREs, one plausible possibility is that Gaf-1/Rip11 may
provide a docking site for
-SNAP as SNAREs do for
-SNAP. If this
were the case, a complex comprising
-SNAP and Gaf-1/Rip11 would be
disassembled in a manner coupled to NSF-mediated ATP hydrolysis. Our
present results clearly showed the requirement of NSF for the
disassembly of the
-SNAP-Gaf-1/Rip11. It is possible that
-SNAP,
in cooperation with NSF, may regulate the function of members of the
Rab11-interacting protein family.
 |
ACKNOWLEDGEMENTS |
We thank K. Mitsui and K. Okada for technical
assistance and Dr. R. Prekeris for critical reading of the manuscript.
We also thank Dr. T. Yoshimori at the National Institute for Basic
Biology, Dr. Y. Takai at Osaka University, and Dr. S. W. Whiteheart at the University of Kentucky College of Medicine for
kindly providing useful materials.
 |
FOOTNOTES |
*
This work was supported in part by Grants-in-aid for
Scientific Research 13680792, 10215205, and 14380339 from the
Ministry of Education, Science, Sports and Culture of Japan, and by ONO Medical Research Foundation.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.: 81-426-77-7496;
Fax: 81-426-76-8866; E-mail: tagaya@ls.toyaku.ac.jp.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M213205200
 |
ABBREVIATIONS |
The abbreviations used are:
NSF, N-ethylmaleimide-sensitive factor;
GST, glutathione
S-transferase;
NEM, N-ethylmaleimide;
SNAP, soluble NSF attachment protein;
SNARE, SNAP receptor.
 |
REFERENCES |
1.
|
Wickner, W.,
and Haas, A.
(2000)
Annu. Rev. Biochem.
69,
247-275[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Lin, R. C.,
and Scheller, R. H.
(2000)
Annu. Rev. Cell Dev. Biol.
16,
19-49[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Whiteheart, S. W.,
Schraw, T.,
and Matveeva, E. A.
(2001)
Int. Rev. Cytol.
207,
71-112[Medline]
[Order article via Infotrieve]
|
4.
|
Mayer, A.
(2002)
Annu. Rev. Cell Dev. Biol.
18,
289-314[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Weber, T.,
Zemelman, B. V.,
McNew, J. A.,
Westermann, B.,
Gmachl, M.,
Parlati, F.,
Söllner, T. H.,
and Rothman, J. E.
(1998)
Cell
92,
759-772[Medline]
[Order article via Infotrieve]
|
6.
|
McNew, J. A.,
Parlati, F.,
Fukuda, R.,
Johnston, R. J.,
Paz, K.,
Paumet, F.,
Söllner, T. H.,
and Rothman, J. E.
(2000)
Nature
407,
153-159[CrossRef][Medline]
[Order article via Infotrieve]
|
7.
|
Wilson, D. W.,
Whiteheart, S. W.,
Wiedmann, M.,
Brunner, M.,
and Rothman, J. E.
(1992)
J. Cell Biol.
117,
531-538[Abstract]
|
8.
|
Söllner, T.,
Whiteheart, S. W.,
Brunner, M.,
Erdjument-Bromage, H.,
Geromanos, S.,
Tempst, P.,
and Rothman, J. E.
(1993)
Nature
362,
318-324[CrossRef][Medline]
[Order article via Infotrieve]
|
9.
|
Hanson, P. I.,
Otto, H.,
Barton, N.,
and Jahn, R.
(1995)
J. Biol. Chem.
270,
16955-16961[Abstract/Free Full Text]
|
10.
|
Whiteheart, S. W.,
Griff, I. C.,
Brunner, M.,
Clary, D. O.,
Mayer, T.,
Buhrow, S. A.,
and Rothman, J. E.
(1993)
Nature
362,
353-355[CrossRef][Medline]
[Order article via Infotrieve]
|
11.
|
Morgan, A.,
Dimaline, R.,
and Burgoyne, R. D.
(1994)
J. Biol. Chem.
269,
29347-29350[Abstract/Free Full Text]
|
12.
|
Barnard, R. J. O.,
Morgan, A.,
and Burgoyne, R. D.
(1997)
J. Cell Biol.
139,
875-883[Abstract/Free Full Text]
|
13.
|
Clary, D. O.,
Griff, I. C.,
and Rothman, J. E.
(1990)
Cell
61,
709-721[Medline]
[Order article via Infotrieve]
|
14.
|
Griff, I. C.,
Schekman, R.,
Rothman, J. E.,
and Kaiser, C. A.
(1992)
J. Biol. Chem.
267,
12106-12115[Abstract/Free Full Text]
|
15.
|
Rice, L. M.,
and Brunger, A. T.
(1999)
Mol. Cell
4,
85-95[Medline]
[Order article via Infotrieve]
|
16.
|
DeBello, W. M.,
O'Connor, V.,
Dresbach, T.,
Whiteheart, S. W.,
Wang, S. S.-H.,
Schweizer, F. E.,
Betz, H.,
Rothman, J. E.,
and Augustine, G. J.
(1995)
Nature
373,
626-630[CrossRef][Medline]
[Order article via Infotrieve]
|
17.
|
Morgan, A.,
and Burgoyne, R. D.
(1995)
EMBO J.
14,
232-239[Abstract]
|
18.
|
Peter, F.,
Wong, S. H.,
Subramaniam, V. N.,
Tang, B. L.,
and Hong, W.
(1998)
J. Cell Sci.
111,
2625-2633[Abstract/Free Full Text]
|
19.
|
Whiteheart, S. W.,
Brunner, M.,
Wilson, D. W.,
Wiedmann, M.,
and Rothman, J. E.
(1992)
J. Biol. Chem.
267,
12239-12243[Abstract/Free Full Text]
|
20.
|
Chen, D.,
Xu, W.,
He, P.,
Medrano, E. E.,
and Whiteheart, S. W.
(2001)
J. Biol. Chem.
276,
13127-13135[Abstract/Free Full Text]
|
21.
|
Nagase, T.,
Ishikawa, K.,
Suyama, M.,
Kikuno, R.,
Hirosawa, M.,
Miyajima, N.,
Tanaka, A.,
Kotani, H.,
Nomura, N.,
and Ohara, O.
(1998)
DNA Res.
5,
355-364[Medline]
[Order article via Infotrieve]
|
22.
|
Prekeris, R.,
Klumperman, J.,
and Scheller, R. H.
(2000)
Mol. Cell
6,
1437-1448[Medline]
[Order article via Infotrieve]
|
23.
|
Prekeris, R.,
Davies, J. M.,
and Scheller, R. H.
(2001)
J. Biol. Chem.
276,
38966-38970[Abstract/Free Full Text]
|
24.
|
Hales, C. M.,
Griner, R.,
Hobdy-Henderson, K. C.,
Dorn, M. C.,
Hardy, D.,
Kumar, R.,
Navarre, J.,
Chan, E. K. L.,
Lapierre, L. A.,
and Goldenring, J. R.
(2001)
J. Biol. Chem.
276,
39067-39075[Abstract/Free Full Text]
|
25.
|
Lindsay, A. J.,
Hendrick, A. G.,
Cantalupo, G.,
Senic-Matuglia, F.,
Goud, B.,
Bucci, C.,
and McCaffrey, M. W.
(2002)
J. Biol. Chem.
277,
12190-12199[Abstract/Free Full Text]
|
26.
|
Lindsay, A. J.,
and McCaffrey, M. W.
(2002)
J. Biol. Chem.
277,
27193-27199[Abstract/Free Full Text]
|
27.
|
Wallace, D. M.,
Lindsay, A. J.,
Hendrick, A. G.,
and McCaffrey, M. W.
(2002)
Biochem. Biophys. Res. Commun.
292,
909-915[CrossRef][Medline]
[Order article via Infotrieve]
|
28.
|
Tagaya, M.,
Wilson, D.,
Brunner, M.,
Arango, N.,
and Rothman, J. E.
(1993)
J. Biol. Chem.
268,
2662-2666[Abstract/Free Full Text]
|
29.
|
Tanaka, M.,
Gupta, R.,
and Mayer, B. J.
(1995)
Mol. Cell. Biol.
15,
6829-6837[Abstract]
|
30.
|
Tani, K.,
Mizoguchi, T.,
Iwamatsu, A.,
Hatsuzawa, K.,
and Tagaya, M.
(1999)
J. Biol. Chem.
274,
20505-20512[Abstract/Free Full Text]
|
31.
|
Whiteheart, S. W.,
Rossnagel, K.,
Buhrow, S. A.,
Brunner, M.,
Jaenicke, R.,
and Rothman, J. E.
(1994)
J. Cell Biol.
126,
945-954[Abstract]
|
32.
|
Nagiec, E. E.,
Bernstein, A.,
and Whiteheart, S. W.
(1995)
J. Biol. Chem.
270,
29182-29188[Abstract/Free Full Text]
|
33.
|
McMahon, H. T.,
and Südhof, T. C.
(1995)
J. Biol. Chem.
270,
2213-2217[Abstract/Free Full Text]
|
34.
|
Hatsuzawa, K.,
Hirose, H.,
Tani, K.,
Yamamoto, A.,
Scheller, R. H.,
and Tagaya, M.
(2000)
J. Biol. Chem.
275,
13713-13720[Abstract/Free Full Text]
|
35.
|
McMahon, H. T.,
Ushkaryov, Y. A.,
Edelmann, L.,
Link, E.,
Binz, T.,
Niemann, H.,
Jahn, R.,
and Südhof, T. C.
(1993)
Nature
364,
346-349[CrossRef][Medline]
[Order article via Infotrieve]
|
36.
|
Ravichandran, V.,
Chawla, A.,
and Roche, P. A.
(1996)
J. Biol. Chem.
271,
13300-13303[Abstract/Free Full Text]
|
37.
|
Haynes, L. P.,
Barnard, R. J.,
Morgan, A.,
and Burgoyne, R. D.
(1998)
FEBS Lett.
436,
1-5[CrossRef][Medline]
[Order article via Infotrieve]
|
38.
|
Meyers, J. M.,
and Prekeris, R.
(2002)
J. Biol. Chem.
277,
49003-49010[Abstract/Free Full Text]
|
39.
|
Cullis, D. N.,
Philip, B.,
Baleja, J. D.,
and Feig, L. A.
(2002)
J. Biol. Chem.
277,
49158-49166[Abstract/Free Full Text]
|
40.
|
Hayashi, T.,
Yamasaki, S.,
Nauenburg, S.,
Binz, T.,
and Niemann, H.
(1995)
EMBO J.
14,
2317-2325[Abstract]
|
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Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.