From the Max Planck Institute for Biophysical Chemistry, Department of Molecular Genetics, D-37070 Göttingen, Germany
Received for publication, December 19, 2000
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ABSTRACT |
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Gyp6p from yeast belongs to the GYP family of
Ypt/Rab-specific GTPase-activating proteins, and Ypt6p is its preferred
substrate (Strom, M., Vollmer, P., Tan, T. J., and Gallwitz, D. (1993) Nature 361, 736-739). We have investigated the
kinetic parameters of Gyp6p/Ypt6p interactions and find that Gyp6p
accelerates the intrinsic GTPase activity of Ypt6p (0.0002 min Monomeric GTPases of the Ras superfamily act as regulators in many
vital cellular processes. They switch their conformation depending on
the nucleotide being bound. Ras and Ras-like proteins bind GDP and GTP
specifically and with high affinity, and they are able to hydrolyze the
bound GTP but with low efficiency. In general, as the switch from the
GTP-bound to the GDP-bound conformation results in the termination of
the functional stimulus by a given GTPase, the acceleration of the slow
intrinsic GTPase activity (often far below 1 min GTPase-activating proteins for Ypt/Rab transport GTPases were first
discovered in yeast (3, 4) and shown to share several conserved
sequence motifs with a variety of proteins from other eukaryotic
species (5). These sequences are localized within the catalytically
active region of the yeast GAPs, Gyp1p and Gyp7p (6), and we refer to
this region as the GYP domain. However, additional sequences C-terminal
of the GYP domain are required for GAP activity (6). Eight yeast
proteins and one mammalian protein containing the GYP domain are known
to be Ypt/Rab-specific GAPs (3, 4,
6-11).2 The length of the
eight GYP family members in yeast ranges from 458 to 950 amino acid
residues. The GYP domain, which in Gyp1p and Gyp7p is localized within
the C-terminal half of the proteins, covers a region of approximately
300 amino acids. The N-terminal halves of both GAPs can be deleted
without affecting the catalytic activity or the substrate specificity
in vitro (6). Therefore, it appears that these sequences
serve other purposes within the cell, such as the interaction with
other cellular components to direct the GAPs to their scene of action.
With 458 amino acids, the Ypt6p-specific Gyp6p (3) is the smallest of
the eight known Ypt/Rab GAPs from yeast and apparently has little
sequence space outside the GAP catalytic domain. This observation and
the fact that its overall sequence deviates more significantly from the
other GAPs prompted us to study the biochemical characteristics of this
protein. We found that short N-terminal or C-terminal deletions
inactivate Gyp6p and that Gyp6p accelerates the intrinsic GTPase
activity of Ypt6p >106-fold and most probably uses a
catalytic arginine, which is in the corresponding position of the
critical arginine identified in Gyp1p and Gyp7p (6, 12).
Cloning Strategies--
All cloning procedures were performed
using standard protocols (13). For construction of the yeast expression
vector pEG(KT)-GYP6, the plasmid pGEX-GYP6 (bearing a BamHI
restriction site 5' adjacent to the ATG start codon and an
EcoRI site directly following the TAA translational stop
codon of GYP6) was digested with EcoRI, treated
with the Klenow fragment, and cleaved with BamHI. The isolated GYP6-containing fragment was ligated into the
BamHI- and XbaI-cleaved vector pEG(KT) (14) after
filling in the overhanging ends of the XbaI cleavage site
with the Klenow enzyme. The C-terminal deletions of Gyp6p were
generated by inserting the following GYP6 sequence-containing fragments into pEG(KT) that are derived from pGEX-GYP6: BamHI-AccI (for Gyp6(1-277)p),
BamHI-SphI (for Gyp6(1-323)p), and
BamHI-EcoNI (for Gyp6(1-382)p). For
N-terminal deletions, a HindIII-EcoRI fragment
(for Gyp6(45-458)p), a BspMI-EcoRI fragment (for
Gyp6(182-458)p), or a BglII-EcoRI fragment (for
Gyp6(209-458)p) was isolated from pGEX-GYP6, and a
BanI-XbaI fragment (for Gyp6(72-458)p) was
isolated from pRS326-GYP6 (3). The fragments were blunt-end inserted
into pEG(KT) in frame with the GST-encoding sequence. Proper expression
of the truncated GST-Gyp6 fusion proteins was verified by Western blot
analysis using anti-GST antisera (Amersham Pharmacia Biotech).
Production and Purification of Proteins--
Ypt6p was produced
in Escherichia coli using the pET vector system (Novagen)
and purified as described previously (15). GST-Gyp6 fusion proteins
were produced in the yeast strain BJ5459 (MATa ura3-52
trp1 lys2-801 leu2 GAP Activity Assays--
GAP activity in crude yeast cell
extracts was determined using the filter assay and
[ Two-hybrid Analysis--
The generation of pAS2-YPT6,
pAS1-YPT6(Q69L), and pACTII-GYP6 has been described previously (17).
The fusion of the Gal4 DNA binding domain (Gal4-BD) with Ypt6(G20S)p
was performed by the insertion of an NcoI/BglII
fragment from pACTII-YPT6(G20S) into
NcoI/BamHI-cleaved pAS2, and the codon exchange
was verified by sequence analysis. Truncated Gyp6 proteins were fused
to the Gal4 transcription activation domain in the two-hybrid vector pACTII. Proper expression of the "bait" and "prey" plasmids in the yeast reporter strain Y190 (18) was controlled by Western blot
analysis using hemagglutinin monoclonal antibody (Roche Molecular Biochemicals). Double transformants expressing both bait and
prey hybrid proteins were analyzed for Site-directed Mutagenesis of GYP6--
Substitution of arginine
to alanine or lysine codons in GYP6 was achieved by a
PCR-based overlap extension method (22) using the following
oligonucleotides as primers (mutated codons are underlined):
R39A, 5'-CTTTAAAGAAAATAGTGCCGGCTGGCTCTGGAA-3' and 5'-TTCCAGAGCCAGCCGGCACTATTTTCTTTAAAG-3'; R155A,
5'-AGATTTGGACCTCTCGGCCATAATGCTTGACGAT-3' and
5'-ATCGTCAAGCATTATGGCCGAGAGGTCCAAATCT-3'; R155K,
5'-GGACCTCTCAAAGATAATGCTTGACG-3' and
5'-CGTCAAGCATTATCTTTGAGAGGTCC-3'; R290A,
5'-AATCTGGCTCATCGCCTGGACAAGGTTGTT-3' and
5'-AACAACCTTGTCCAGGCGATGAGCCAGATT-3'; R298A,
5'-GGACAAGGTTGTTATTTTTGGCCGAATTACCCTTAAAAT-3' and
5'-ATTTTAAGGGTAATTCGGCCAAAAATAACAACCTTGTCC-3'.
Catalytic Properties of the Ypt6-GAP Gyp6p--
We had previously
shown that bacterial expression of full-length Gyp6 protein allowed us
to identify its activity as a Ypt/Rab-specific GAP (3), but large scale
production of the protein in E. coli failed. Likewise, we
were unable to produce a GST-Gyp6 fusion protein in E. coli
in reasonable quantity. Therefore, we expressed the GST-Gyp6p fusion in
a proteinase-deficient yeast strain under control of the strong
galactose-inducible GAL10 promoter. As thrombin cleavage of the
affinity-purified fusion protein resulted in a significant loss of GAP
activity, the biochemical analyses were performed with GST-Gyp6p
fusions that were >80% pure. GAP activity was determined with varying
amounts of the fusion protein and a 10-100-fold excess (20 µM) of GTP-bound Ypt6p. From the initial rates of GTP
hydrolysis determined by high pressure liquid chromatography-based quantification of GTP and GDP, a specific activity of 72.2 (± 4.8)
units/nmol GST-Gyp6p was calculated where one unit of GAP was defined
as the hydrolysis of 1 nmol of Ypt6p-bound GTP in 1 min at 30 °C.
This value compares well with specific activities that we recently
determined for two other yeast Ypt/Rab GAPs (6).
For further characterization of the Gyp6p-Ypt6p interaction, the
Km and kcat values were
determined from single time curves using the integrated
Michaelis-Menten equation (23) as described for the analysis of the
catalytic properties of Gyp1p, Gyp7p, and Gyp3p (6, 9). In a
representative experiment using 100 nM GST-Gyp6p and an
initial substrate concentration of 200 µM Ypt6p-GTP, we
determined Km = 592 µM and
kcat = 18.8 s Impairment of GAP Activity by Single Point and Truncation
Mutations--
Within the GYP family of yeast Ypt/Rab-GAPs, Gyp6p is
an exception in that it lacks a larger N-terminal sequence segment
preceding the GYP domain (Fig. 1). The
GYP domain of the Ypt6-GAP with its six elements A-F, related in
primary sequence among all GYP family members (5, 12), shares with that
of Gyp1p and Gyp7p four arginine residues in equivalent
positions. Two of these residues, Arg-39 and Arg-155, of Gyp6p
are strictly conserved in all known Ypt/Rab-GAPs of the GYP family. We
have shown by mutational analysis that the conserved arginine in
sequence segment B of the GYP domain (Fig. 1) is required for the
catalytic activity of Gyp1p and Gyp7p (6). We generated GST-Gyp6 fusion
proteins with alanine substitutions for the four arginines in position
39 (motif A), 155 (motif B), and 290 and 298 (motif F) in addition to a mutant GAP with the conservative
R155K exchange. From kinetic analyses under standard conditions (Fig.
2), we observed that all substitutions
led to significant inhibition of Gyp6p GAP activity (Table
II). Importantly, both the R155K and the
R155A substitution resulted in an apparently complete inactivation of
the Gyp6 mutant proteins.
Because we were unable to achieve GST-Gyp6p concentrations high enough
to measure the Km and kcat
values for the two mutant proteins, a distinction between an impairment
of catalysis and that of substrate affinity was attempted using the
two-hybrid system. To test for substrate protein binding, Gyp6p and
Ypt6p were fused to the Gal4 transcription activation domain and
Gal4-BD, respectively, and protein-protein interactions were identified by monitoring
Because the large N-terminal segments preceding the GYP domain in Gyp1p
and Gyp7p could be deleted without reduction of the catalytic activity
(6), we addressed the question of whether Gyp6p contained N-terminal or
C-terminal sequences dispensable for catalytic activity or substrate
GTPase binding. Successive N-terminal and C-terminal deletions were
created using available restriction enzyme cutting sites of the
GYP6 protein-coding region. GST fusions of the truncated
Gyp6 proteins were expressed in yeast (Fig.
3), and their apparent GAP activity was
assessed in cellular extracts with [
Substrate binding of Gyp6p truncation mutants was assessed by
two-hybrid analyses to determine whether the lack of GAP activity was
the result of an impaired physical GAP/GTPase interaction. Truncated
Gyp6 proteins fused to Gal4 transcription activation domain were
coexpressed with Gal4-BD-Ypt6p or Gal4-BD-Ypt6(Q69L)p, expression was
confirmed by Western blotting analysis, and the interaction was
analyzed using the X-gal filter test (Fig.
4B). The expression level of
the truncated GAPs was comparable to that of full-length Gyp6p (data
not shown). Very weak interaction of Ypt6(Q69L)p but not wild-type
Ypt6p was detected with Gyp6(45-458)p. No interaction was observed
with two larger N-terminal deletions or with the C-terminal truncation
mutant Gyp6(1-325)p, which lacked most of the sequences distal of the
GYP domain (Fig. 1). These results suggest that the GYP domain as well
as sequences C-terminal of the GYP domain contribute to
efficient substrate binding.
Our recent studies have shown that the deletion of extended
regions, which are located N-terminal of the catalytic domain of most
GYP family members, does not inhibit GAP activity nor does it affect
substrate specificity (6).3
In Gyp1p and Gyp7p, this GAP-dispensable region amounts to 39 and 48%,
respectively, of the total length of the protein. In fact, the
catalytically active fragment of both proteins is more active than the
full-length GAPs. As gyp1 (7), gyp6 (3), gyp7 (8), and other Ypt-GAP null mutants are phenotypically inconspicuous in complete growth media, it is not an easy task to elucidate the function(s) of the N-terminal domains in
vivo. We have argued (6) that because of the low affinity of
Ypt/Rab-GAPs to their substrate GTPases, high concentrations of the
GAPs would be required at those membranes where they are likely to act
and that the N-terminal extensions of the GAPs might be required for their recruitment to specific cellular locations. If this were the
case, Gyp6p without a fragment of appreciable length preceding the GYP
domain would need to employ other part(s) of the molecule for
localization purposes.
All of the N-terminal and C-terminal truncations of Gyp6p we have
described here significantly inhibited or even abolished GAP activity.
The shortest of the truncations, Gyp6(45-458)p and Gyp6(1-382)p, are
of special interest. The deletion of the N-terminal 44 amino acids
included most of the 15 amino acid-long sequence motif A with Arg-39
and Trp-43, two residues strictly conserved in all GYP family members
(Fig. 1). This mutant not only lost GAP activity but also its ability
to bind its substrate GTPase Ypt6p as shown by two-hybrid analysis. The
latter finding strengthens the argument, derived from our recently
solved x-ray structure of the Gyp1p catalytic domain, that the
conserved arginine and tryptophan residues in motif A contribute to the
stabilization of the tertiary structure of the GAP domain and
presumably to the formation of the GTPase-binding epitope (12). The
C-terminal truncation mutants of Gyp1p and Gyp7p, which we previously
studied and found to be catalytically inactive, terminated only 31 and 17 amino acids, respectively, distal of the motif F of the GYP domain.
But in this study, we had not addressed the question of whether the
truncated GAPs were still able to bind their substrate proteins. The
work with Gyp6p now shows that a segment of 13 amino acid residues
C-terminal of the GYP domain (truncation mutant Gyp6(1-323)p) is not
sufficient to allow binding to the substrate GTPase. Even Gyp6(1-382)p
with 72 amino acids following the GYP domain was inactive most probably
because of its deficiency for substrate binding. The crystal structure
of Gyp1p and the proposed Gyp1p·Ypt51p complex (12) suggests
that at least one Apart from its exceptional N terminus among the GYP family members,
Gyp6p, which for technical reasons had to be analyzed as a GST fusion
protein, shares similar biochemical properties with other
Ypt/Rab-specific GAPs studied. It accelerates the low intrinsic
GTP hydrolysis rate very potently but displays very low affinity
(Km > 500 µM) for its preferred
substrate Ypt6p. In fact, with a 5 × 106-fold
acceleration of the basic GTPase activity of Ypt6p, Gyp6p is the most
potent of the Ypt/Rab-GAPs that we have analyzed so far. Although the
substitution with alanine of four arginine residues within the shared
sequence motifs of the GYP domain led to a significant loss of Gyp6p
GAP activity, only the strictly conserved arginine in position 155 proved to be essential for GAP activity. Importantly, as we could
demonstrate for Gyp6(R155A) in a two-hybrid analysis, substitutions of
Arg-155 do not interfere with GAP/GTPase interaction. This finding is
further evidence for the suggestion, based on our mutational (6, 9, 11)
and structural (12) investigations, that this particular arginine is
directly involved in catalysis.
1) by a factor of 5 × 106 and that
they have a very low affinity for its preferred substrate (Km = 592 µM). Substitution with
alanine of several arginines, which Gyp6p shares with other GYP family
members, resulted in significant inhibition of GAP activity.
Replacement of arginine-155 with either alanine or lysine abolished its
GAP activity, indicating a direct involvement of this strictly
conserved arginine in catalysis. Physical interaction of the
catalytically inactive Gyp6(R155A) mutant GAP with Ypt6 wild-type and
Ypt6 mutant proteins could be demonstrated with the two-hybrid system.
Short N-terminal and C-terminal truncations of Gyp6p resulted in a
complete loss of GAP activity and Ypt6p binding, showing that in
contrast to two other Gyp proteins studied previously, most of the 458 amino acid-long Gyp6p sequence is required to form a three-dimensional
structure that allows substrate binding and catalysis.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1) must
be an important device to regulate the activity of the regulator.
GTPase-activating proteins
(GAPs),1 specific for Ras,
Rho, and Ypt/Rab family members that are able to activate the
hydrolysis rate of GTPase-bound GTP by several orders of magnitude,
have been isolated from many eukaryotes and found to be important for
the functional cycle of GTPase (for review, see Refs. 1 and
2).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
1 his3
200
pep4::HIS3 prb1
1.6R can1 GAL)
(Yeast Genetic Stock Center, University of California at Berkeley) and
purified as described previously for the preparations of GST-Gyp7p (8).
To get active protein, buffers used had to be free of CHAPS. Yeast cell
lysis was done in buffer consisting of 50 mM Tris-HCl, pH
7.5, 5 mM EDTA, 100 mM KCl, 1 mM
Pefabloc and 1× complete proteinase inhibitors (Roche Molecular Biochemicals), column washes in 50 mM Tris-HCl pH 7.5, 5 mM EDTA, 1 M KCl. GST-Gyp6p was eluted from
glutathione-Sepharose at 4 °C in 50 mM Tris-HCl, 20 mM reduced glutathione, pH 7.5. Concentration and purity of
the GST fusion proteins were determined as described previously
(6).
32P]GTP-loaded Ypt6p (4). The high pressure liquid
chromatography-based quantitative GAP assay used for analysis of
purified proteins and the evaluation of the Gyp6p/Ypt6p interaction
using the integrated Michaelis-Menten equation were performed as
described recently in detail (6, 16).
-galactosidase
activity by the X-gal filter assay as described previously (19).
Quantification of the
-galactosidase activity in liquid cultures
using o-nitrophenyl-
-D-galactosidase was
performed as described by Guarente (20) and evaluated according to
Miller (21), units = A420 /
(t × V × A600)
where t is the reaction time (in min) at 30 °C, and
V is the volume (in ml) of the yeast culture used in the assay.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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1 for GST-Gyp6p.
Given the slow intrinsic GTP hydrolysis rate of Ypt6p (0.0002 min
1), this means a maximal acceleration of
5.6 × 106-fold. Thus, compared with the catalytic
properties of other GAPs for Ypt-GTPases, Gyp6p seems to bind its
substrate with very low affinity but causes the highest maximal
acceleration of GTP hydrolysis measured so far for a Ypt/Rab-GAP
(Table I).
Catalytic properties of Ypt-specific GTPase activating proteins
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Fig. 1.
Schematic representation of the structural
makeup of yeast Ypt/Rab-specific GAPs. The location of the
GYP domain with the related sequence segments A-F
highlighted in red is compared between Gyp6p and
Gyp1p. Amino acid residues strictly conserved in segments A
and B of all GYP family members are shown; the arginine
likely to be involved in catalysis is highlighted in
yellow. Two mutant Gyp6 proteins (with the shortest
C-terminal and N-terminal deletions) that are catalytically inactive in
Ypt6 GTPase binding are shown at the top. Relevant amino
acid residues are numbered.
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Fig. 2.
Effect of arginine substitutions on the
catalytic activity of Gyp6p. 1 µM of either
wild-type (wt) or mutant Gyp6p fused to GST was incubated
with 20 µM GTP-loaded Ypt6p at 30 °C. At the time
points indicated, aliquots of the incubation mixtures were shock
frozen, and GTP and GDP concentrations were determined by high pressure
liquid chromatography analysis. Note that R155A and R155K substitutions
only led to an apparent complete loss of Gyp6p catalytic
activity.
Activity of GST-Gyp6 proteins with arginine substitutions
-galactosidase activity in a reporter yeast strain transformed with both expression plasmids. Expression of wild-type and
mutant proteins was verified by Western blotting analysis using
appropriate antibodies. Although GAP/GTPase interactions must be
extremely short-lived because of the fast GTP hydrolysis reaction,
Gyp6p-interaction with its substrate GTPase Ypt6p could be demonstrated
(Table III). We also observed a weaker
interaction of the GAP with Ypt6p mutated in the P-loop (G20S
substitution), and as expected, a considerable enhancement of GAP
interaction with Ypt6(Q69L)p, a mutant GTPase that like other Ras
superfamily members with the same substitution in the equivalent
position, has a significantly reduced intrinsic GTPase activity (Fig.
4A). When the catalytically inactive Gyp6(R155A) mutant GAP
was analyzed with respect to its binding to wild-type and mutant Ypt6
GTPases, we found that the GAP/GTPase interactions were significantly
stronger than they were with the wild-type Gyp6 protein. This
was especially apparent with the P-loop mutant Ypt6(G20S)p (Table III).
From this study, it follows that substitutions of Arg-155 in Gyp6p
affect the catalytic activity of GAP rather than its substrate
binding.
Interaction of Gyp6p and Ypt6p in the two-hybrid system
-32P]GTP-loaded
Ypt6p using a filter assay. Whereas cell extracts containing
full-length GST-Gyp6p generally resulted in the hydrolysis of >80%
Ypt6p-bound GTP within 10 min at 30 °C, extracts containing truncated GST-Gyp6 fusion proteins exhibited only background
activity in at least three independent experiments. This finding shows that GAP activity of Gyp6p is already lost or significantly reduced in
mutant proteins lacking either the N-terminal 44 amino acid residues
(including most of the segment A sequence of the GYP domain) or the
C-terminal 76 residues (still leaving intact a block of 72 amino acids
distal of the GYP domain) (see Fig. 1).
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Fig. 3.
N-terminal and C-terminal truncated Gyp6
proteins produced in yeast. Wild-type Gyp6p and its truncated
versions were synthesized in a proteinase-deficient yeast strain from a
multi-copy plasmid under the control of the galactose-inducible GAL10
promoter. Total cellular protein was separated by SDS-polyacrylamide
gel electrophoresis, transferred to nitrocellulose, and GST and
GST-Gyp6 fusion proteins were identified immunologically with anti-GST
antibodies. Numbers in parentheses indicate the
N-terminal and C-terminal Gyp6p residues of the truncation
mutants.
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Fig. 4.
Two-hybrid interactions of Gyp6p and its
substrate GTPase Ypt6p. A, the Y190 reporter
strain was transformed with pACTII-GYP6 as prey in combination with
empty pAS2, pAS2-YPT6 (wild-type), pAS1-YPT6(G20S), or pAS1-YPT6(Q69L)
as baits. B, prey constructs encoding truncated Gyp6 hybrid
proteins were combined with either pAS2-YPT6 wild-type
(Ypt6p(wt)) or pAS1-YPT6(Q69L). -Galactosidase
activity of double transformants grown on agar plates for 2 days was
tested by the X-gal filter assay. Production of all fusion proteins was
verified by Western blot analysis with total protein of the
transformants.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical region located approximately 100 amino
acid residues C-terminal of the GYP domain could contribute to GTPase
binding. The C-terminal region distal of the GYP domain of different
GYP family members is at least 150 amino acid residues long, but the
primary sequences are quite divergent. This work clearly indicates that
a significant part of this region is required for the overall
architecture of an active GAP and for the binding of the substrate GTPases.
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ACKNOWLEDGEMENTS |
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We thank Rita Schmitz-Salue, Ursula Welscher-Altschäffel, and Hans-Peter Geithe for technical help and Ingrid Balshüsemann for secretarial assistance.
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FOOTNOTES |
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* This work was supported by the Max Planck Society and by grants from the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie (to D. G.).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.
Present address: Dept. of Cell and Virus Genetics, Heinrich Pette
Inst., D-20251 Hamburg, Germany.
§ To whom correspondence should be addressed. Tel.: 49-551-201-1496; Fax: 49-551-201-1718; E-mail: dgallwi1@gwdg.de.
Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.M011451200
2 S. Albert, A. DeAntoni, and D. Gallwitz, unpublished observations.
3 S. Albert, A. DeAntoni, and D. Gallwitz, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
GAP, GTPase-activating protein;
GST, glutathione S-transferase;
X-gal, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside;
Gal4-BD, Gal4 DNA binding domain;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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