From the Department of Biochemistry, McMaster University, Hamilton, Ontario L8N 3Z5, Canada
Received for publication, March 3, 2003 , and in revised form, May 13, 2003.
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ABSTRACT |
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INTRODUCTION |
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The concerted action of GTPases ensures that the targeting step and nascent
chain transfer step are unidirectional processes
(5). SRP54 and one of the
subunits of the SRP receptor, SR, bind GTP in a cooperative manner
(6). Binding of GTP by SRP54
and SR
increases the affinity of these proteins for one another,
thereby maintaining a direct physical link between the ribosome-nascent chain
and the ER membrane (7,
8). Sequence comparisons
revealed that the SR
and SRP54 GTPases define a specific subfamily of
GTPases conserved in prokaryotes, yeast, and mammals that is now referred to
as the SRP family of GTPases
(9,
10). The third GTPase that
appears to be involved in regulating translocation is SR
. It has been
proposed that release of SRP from the nascent chain, and subsequent transfer
of the nascent chain to the translocon, is controlled by SR
(11).
Unlike SRP54 and SR, SR
shares significant homology within the
GTP binding consensus sequences, or G boxes, of Ras-type GTPases
(12). Structural analysis
indicates that SR
also bears significant structural homology to Ras-type
GTPases (13). However, it
differs from other Ras-type GTPases in two respects. First, although other
Ras-type GTPases require prenylation at the carboxyl terminus to enable a
reversible interaction with membranes
(14), SR
is permanently
integrated into the ER membrane by an amino-terminal transmembrane domain.
Second, SR
contains a cysteine within the G1 GTPase consensus sequence
where most Ras-type GTPases contain a glycine. The other exception is members
of the Arf family of GTPases that all contain an aspartic acid at this
position. The identity of this amino acid appears to be crucial to the
activity of Arf and Ras GTPases
(1517),
which raises the possibility that SR
differs functionally from both
Arf-like and other Ras-type GTPases.
The search for protein factors that influence the activity of SR has
led to the observation that a factor associated with ribosomes possesses
measurable GAP activity and may also function as a guanine nucleotide
dissociation factor for SR
. Incubation of ribosome-nascent chain
complexes with either the SR
/SR
dimer or a proteolysis product of
the dimer lacking the SR
GTPase (SR
) both increases the
GTPase activity of SR
and decreases the affinity of SR
for
nucleotides (18). The
influence of the ribosome on SR
suggests a direct physical contact
between the two. Supporting this prediction, cross-linking experiments have
revealed an interaction between SR
and a protein component of the
ribosomal 60 S subunit (11).
Whether this ribosomal protein is responsible for the observed GAP/guanine
nucleotide dissociation factor activity is still unresolved.
In addition to proposed roles in nascent chain transfer and SRP release,
SR also anchors SR
to the ER membrane via a tight physical
interaction between the SR
GTPase domain and an amino-terminal domain of
SR
(19,
20). This interaction is
influenced by the nucleotide-bound status of SR
. Generation of empty
SR
by gel filtration of an XTP binding mutant of SR
, which has a
decreased affinity for GTP, abolishes the SR
/SR
interaction.
Replenishing the reaction mixture with xanthosine 5'-diphosphate or XTP
restores dimer formation, with XTP having a greater effect. Deletion of any
part of the SR
core GTPase also abolishes the interaction with SR
(21).
All data gathered to date on the function of SR have been obtained in
the context of a heterodimer. Attempts to isolate the SR
GTPase for
study have involved proteolytic treatment of SR with trypsin or elastase to
specifically digest SR
. This method releases the GTP binding domain of
SR
from SR
but leaves an amino-terminal domain of SR
bound
to SR
(10,
22). Therefore, there are no
data on the properties of SR
as an isolated GTPase. To address this
issue directly we have expressed and purified from Escherichia coli a
soluble version of SR
, termed SR
TM.
Isolated SRTM has no detectable GTPase activity, and most does
not exchange GTP in vitro. The small fraction (36%) of
SR
TM that does bind exogenous GTP undergoes a conformational
change that can be detected by fluorescence spectroscopy. A direct interaction
between SR
TM and the ribosome is confirmed, and the influence of
the ribosome on the SR
GTPase is examined. Our results suggest that
SR
GTPase function is unlike other Ras-type GTPases.
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EXPERIMENTAL PROCEDURES |
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Plasmid pMAC1277 encodes SRTM fused to an amino-terminal His
tag and enterokinase (EK) cleavage site. This plasmid was assembled in two
steps. First pMAC701, encoding SR
md fused to an aminoterminal
His tag and EK cleavage site, was generated by removing the SR
coding
sequence from pMAC455 by digestion with BglII and KpnI and
inserting it into pRSETB (Invitrogen) digested with the same enzymes. The
sequence encoding SR
TM was then excised from pMAC853 using
NcoI and EcoRI and inserted into pMAC701 digested with
NcoI and EcoRI, thereby replacing the coding region for
SR
md with that for SR
TM.
Plasmid pMAC1623 encoding SRC71G
TM fused to an
amino-terminal His tag and EK cleavage site was generated from pMAC1277 by the
method described in Ref. 23.
Briefly, the entire plasmid was amplified by PCR using oligo958
(ATGGGCCCCTCGGCAACTCTGGGAAAAC; desired mutation in bold) and oligo959
(ATGGGCCCCAACAAGAACAGCTCT). The product was then digested with ApaI,
and the 3' over-hanging ends were blunted by incubation with the Klenow
fragment of DNA polymerase, and the linear DNA was circularized by ligation
with T4 DNA ligase.
Plasmid pMAC1624 encodes SRC71D
TM fused to an
amino-terminal His tag and EK cleavage site, under the control of a T7
promoter. To generate this plasmid pMAC1277 was amplified by PCR using
oligo960 (ATGGGCCCCTCGACAACTCTGGGAAAA; desired mutation in bold) and
oligo959. The PCR product was digested with ApaI and end-repaired and
ligated as above.
Plasmid pMAC1278 encodes SRXTP
TM fused to an
amino-terminal His tag and EK cleavage site. SR
XTP
TM
was excised from pMAC1083 with NcoI and EcoRI and inserted
into pMAC701 digested with NcoI and EcoRI, replacing
SR
md with SR
XTP
TM.
Plasmid pMAC1637 encodes SRC71D
TM fused to a
carboxyl-terminal His tag. SR
C71D
TM was amplified from
pMAC1624 using oligo-203 (CATGCCATGGCTAAGTTCATCCGGAGCAGA) and oligo976
(AGAATTCAATGATGATGATGATGATGGGCGATTTTAGCCAGCCAC) and digested with
NcoI and EcoRI. The digested fragment was inserted into
pET16b (Novagen) digested with NcoI and EcoRI.
Protein PurificationPlasmids encoding either
His-SRTM or His-SR
C71D
TM were expressed
in the salt-inducible BL21SI strain by addition of NaCl to 300 mM
final concentration for 2 h. All purification steps were carried out at 4
°C. Cell pellets were washed once in 50 mM
Na2HPO4, pH 8.0, 1 mM phenylmethylsulfonyl
fluoride and resuspended in lysis buffer (50 mM
Na2HPO4, pH 8.0, 500 mM NaCl, 5 mM
MgOAc2, 1 mM phenylmethylsulfonyl fluoride, 10% glycerol
(v/v)). Cells were lysed in a pressure cell, DNA was precipitated with 0.15%
polyethylenamine, and lysate was centrifuged at 18,000 x g for
20 min in a Beckman JA-20 rotor. The lysate was further clarified by
centrifuging at 110,000 x g for 1 h in a Beckman Ti50.2 rotor
prior to loading on Ni-NTA-agarose (Qiagen) equilibrated in 50 mM
Na2HPO4, pH 8.0, 300 mM NaCl, 5 mM
MgOAc2, 10% glycerol. The column was washed in 10 volumes of
equilibration buffer, and His-SR
was eluted with equilibration buffer +
50 mM imidazole. Protein containing fractions were detected by BCA
assay (Pierce), pooled, and dialyzed overnight in 40 mM Tris-OAc,
pH 7.8, 300 mM NaCl, 5 mM MgOAc2, 1
mM DTT, 25% glycerol. Dialysate was diluted with 6 volumes of 40
mM Tris-OAc, pH 7.8, 5 mM MgOAc2, 1
mM DTT, 25% glycerol to reduce the NaCl concentration and loaded
immediately onto CM-Sepharose equilibrated in 40 mM Tris-OAc, pH
7.8, 50 mM NaCl, 5 mM MgOAc2, 1 mM
DTT, 25% glycerol. The column was washed with 10 volumes of equilibration
buffer, and His-SR
was eluted in a single step in SR
elution
buffer (equilibration buffer + 100 mM NaCl). Protein containing
fractions were detected by Bradford assay (Bio-Rad) and pooled. Protein
concentration was determined by absorbance at 280 nm as described
(24). Protein was frozen in
small aliquots at 80 °C; material used for functional studies was
thawed once and discarded.
ImmunoprecipitationProteins were synthesized in vitro, quantified, and immunoprecipitated as described previously (21).
HPLC Analysis of Bound Nucleotide10 nmols of SR were
diluted to 250 µl in SR
elution buffer. An equal volume of 8
M urea, 20 mM Tris-OAc, pH 7.8, 100 mM NaCl
was added, and the sample was incubated at 37 °C for 30 min. The sample
was centrifuged through a 5-kDa cutoff filter (Millipore), and the filtrate
was added to a Bakerbond QUAT 5-µm HPLC column (J. T. Baker Inc.) in 25
mM triethylamine bicarbonate, pH 7.2. Nucleotide was eluted from
the column with a 5100% gradient of triethylamine bicarbonate. Samples
were analyzed with 32Karat, version 3.0 software (Beckman), and nucleotide was
quantified by calculating the area under the curve and comparing to a standard
curve of GTP or GDP. The recovery of nucleotides in these experiments (90%)
was determined by adding a known amount of GMP as an internal control.
Fluorescence Experiments300 nM SR was
incubated with 500 nM 2' (or
3')-O-(N-methylanthraniloyl)GTP (mant-GTP) in 50
mM Tris-Cl, pH 7.6, 150 mM NaCl, 5 mM
MgOAc2, 2 mM DTT, and 10% glycerol in a 1-cm path length
quartz cuvette. All measurements were taken with a fluorometer equipped with
an 815 photomultiplier detection system (PTI, London, Ontario, Canada) with a
2-nm excitation slit width and a 2-nm emission slit width and compiled with
Felix, version 1.4 software (PTI). Samples were excited at 280 or 295 nm, and
emission spectra were obtained by scanning from 300 to 500 nm in 2-nm
increments with an integration time of 0.2 s per data point. Emission spectra
were corrected by subtracting a buffer blank, and peak values were manually
selected for further calculation. All calculations and data plots were
performed within MS Excel 2002.
Filter Binding100 pmols of SR (1 µM)
were incubated at the specified temperatures with 10 µM GTP
including 25% [3H]GTP (specific activity 31 Ci/mmol) in 50
mM Tris-OAc, pH 7.8, 200 mM NaCl, 5 mM
MgCl2, 10% glycerol, 2 mM DTT. At the appropriate time
points samples were withdrawn and diluted to 2 ml in ice-cold filter binding
buffer (20 mM Tris-OAc, pH 7.8, 200 mM NaCl, 5
mM MgCl2, 10 mM NH4Cl). Samples
were applied to prewashed nitrocellulose discs (What-man), and the discs were
washed with 3 x 3-ml filter binding buffer in a Millipore 1225
filtration sampling manifold (Millipore). Discs were dried, and bound
nucleotide was quantified in a scintillation counter.
Nucleotide ExchangeNucleotide exchange reactions were
performed as described previously
(25,
26). Briefly,
SRTM (1 µM) was incubated with 20 µM
GTP including 0.2 µM [
-32P]GTP for 10 min at
30 °C in final buffer conditions containing 20 mM Tris-Cl, pH
7.6, 6 mM MgCl2, 10 mM EDTA, 1 mM
DTT, 10% glycerol. After 10 min MgCl2 was added to a concentration
of 20 mM. The extent of nucleotide exchange was quantified by
filter binding. To prepare SR
TM for GTPase assays, free
nucleotide was separated from bound nucleotide by repurifying
SR
TM on CM-Sepharose.
UV Cross-linking5 µM SR was incubated
with 0.5 µM [
-32P]GTP and the indicated
concentration of unlabeled GTP in cross-linking buffer (50 mM
Tris-OAc, pH 7.8, 150 mM KOAc, 5 mM MgOAc2, 2
mM DTT) for 20 min on ice, followed by 5 min at 24 °C.
Reactions were placed into a plastic weight boat on a chilled metal block and
irradiated with UV light at 5000 microwatts/cm2 for 5 min. Samples
were precipitated with trichloroacetic acid and washed in ethanol:ether (1:1)
to remove free nucleotide, resolved by SDS-PAGE, and analyzed using a
PhosphorImager.
GTPase Assay40 nM nucleotide-bound SR or
5.0 A260 units/ml ribosomes was incubated with 83.5
nM [
-32P]GTP in GTPase buffer (50 mM
Tris-OAc, pH 7.8, 150 mM KOAc, 5 mM MgOAc2, 2
mM DTT) at 24 °C. At the indicated time points samples were
removed and quenched by adjusting the EDTA concentration to 50 mM
on ice. Samples were spotted onto polyethylenamine cellulose TLC plates and
resolved in 0.375 M KH2PO4, pH 3.5, for 1 h.
Plates were dried and exposed to a PhosphorImager screen for quantitative
analysis. To generate the Lineweaver-Burke plot reactions were supplemented
with cold GTP to concentrations up to 5 µM, and the reaction was
monitored using hydrolysis of [
-32P]GTP to estimate
hydrolysis of all GTP.
To assess the effect of ribosomes on the SR GTPase, 10 nM
[
-32P]GTP-loaded SR
TM and 20 nM 80 S
ribosomes were incubated at 24 °C in GTPase buffer. Samples were removed
at the indicated time points and quenched with 50 mM EDTA, and
[
-32P]GTP was resolved from 32Pi by
TLC.
Ribosome Binding ExperimentsCanine pancreatic ribosomes and
wheat germ RNCs were prepared as described elsewhere
(11,
18) and stored at a
concentration of 100 A260 units/ml in 25 mM
HEPES-KOH, pH 7.6, 5 mM MgOAc2, 150 mM KOAc, 1
mM DTT (+1mM cycloheximide for RNCs). 5 µM
SR was incubated with 20 A260 units/ml ribosomes or
RNCs for 1 h at 24 °C in the above buffer and then added to the top of 30
ml of linear 0.31.2 M sucrose gradients. The gradients were
centrifuged in a SW28 rotor for 16 h at 48,000 x g. 1-ml
fractions were collected by bottom puncture and protein precipitated with
trichloroacetic acid, resolved by SDS-PAGE, and analyzed by Western blotting
using an antibody directed against SR
.
Data AnalysisAll data analysis was performed using Sigmaplot 8.02.
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RESULTS |
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In an attempt to identify the importance of the cysteine in the function of
SR two point mutants at this site were generated. The first,
SR
C71D
TM, converts the cysteine to an aspartic acid,
converting SR
into an Arf family GTPase. The second,
SR
C71G
TM, converts the cysteine to a glycine to
resemble other Ras-type GTPases. Binding of SR
and SR
mutants to
SR
was assayed by coprecipitation. SR
, wild-type SR
, both
cysteine point mutants, and another GTPase point mutant,
SR
XTP
TM, shown previously to switch the nucleotide
binding preference from GTP to XTP
(11,
21), were synthesized in
vitro in a rabbit reticulocyte lysate system. Nucleotides were removed
from some samples by gel filtration (GTP) and separate reactions
containing equimolar amounts of SR
and each of the SR
variants
were incubated together to allow complex formation. Complexes were
immunoprecipitated with an antibody against SR
(Fig. 1). All of the SR
molecules bound SR
in the presence of nucleotides contributed by the
translation mix. As reported previously
(21), binding of SR
to
the XTP-preferring version of SR
was greatly reduced in the absence of
nucleotide, because the reduced affinity of SR
XTP
TM
for GTP allows this SR
variant to be emptied by gel filtration. Binding
of either cysteine point mutant to SR
was unaffected by nucleotide
depletion demonstrating that, despite the mutation in the G1 box, these
mutants retain SR
binding activity under conditions that serve to empty
SR
XTP
TM.
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To examine the isolated SR GTPase in greater detail, a recombinant
protein consisting of the cytoplasmic portion of SR
fused to an
amino-terminal hexahistidine tag (His6) was expressed in E.
coli. The protein (SR
TM) was purified to apparent
homogeneity in two steps involving Ni-NTA-agarose and CM-Sepharose
(Fig. 2a). Gel
filtration analysis of the purified product confirmed that SR
TM
is a monomer in solution (data not shown). One of the cysteine point mutants,
SR
C71D
TM, was expressed with a carboxyl-terminal
His6 tag and purified using conditions identical to those used to
purify SR
TM (Fig.
2b).
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GTPases are generally purified in the GDP-bound form. This holds true for
both tissue-derived proteins and recombinant proteins that lack GAP homologues
in E. coli
(2830).
Therefore, we expected that SR purified from E. coli would be
GDP-bound. To identify and quantify the nucleotide that copurified with
SR
TM, 10 nmols of SR
TM or
SR
C71D
TM were denatured in 4 M urea to
release the bound nucleotide into solution. The protein was removed by
filtration, and the released nucleotide was analyzed by HPLC and compared with
standards of GTP and GDP examined in parallel
(Fig. 3). The retention times
for GDP and GTP on the HPLC column were 7.9 and 9.6 min, respectively
(Fig. 3a).
Surprisingly, the supernatant from denatured SR
contained a single major
peak that eluted at 9.6 min, indicating the presence of GTP. A smaller peak
was detected at 7.9 min, corresponding to a small amount of GDP
(Fig. 3b). Calculating
the area under the curves and comparing these values against values obtained
from GTP and GDP standards and correcting for 10% loss (measured using GMP as
an internal standard) revealed that 72% of SR
TM contains bound
GTP whereas only 2.2% was bound to GDP. Similarly, 71% of purified
SR
C71D
TM contains GTP, and 2.8% was bound to GDP.
Therefore both wild-type SR
and the GTPase point mutant remain bound to
GTP throughout purification. The remaining 26% is not bound to nucleotide.
This population of SR
did not bind to GTP in the timescale expected of
an active empty GTPase (31,
32) (see Figs.
4 and
5). Therefore we presume that
this population consists of SR
that has become structurally unstable in
the absence of bound GTP, and the resulting loss of conformation prevented the
uptake of exogenous GTP. A structurally unstable empty state is a common
feature of Ras-type GTPases
(31,
33,
34). Addition of 10
µM GTP to the buffers used during purification did not decrease
the percentage of empty SR
(data not shown), suggesting that this
population does not arise from dissociation of nucleotide during
purification.
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Two methods were used to determine what fraction of recombinant SR is
able to bind to or exchange bound GTP for exogenous GTP. The first method
measured the ability of aromatic amino acids within SR
to transfer
energy to a fluorescent GTP analogue, mant-GTP
(Fig. 4). Resonance energy
transfer (RET) can be measured by monitoring the decrease in fluorescence
output of an excited donor molecule (aromatic amino acids) and concomitant
increase in acceptor molecule (mant) fluorescence
(Fig. 4a). Mant
fluorescence did not change in a control cuvette lacking protein, nor was
there an increase in mant fluorescence attributable to binding to SR
when the dye was excited directly at 350 nm (data not shown). Therefore, the
increase in mant fluorescence arises solely from RET between the mant
fluorophore and aromatic side chains within SR
, including a tryptophan
near the carboxyl terminus. We monitored GTP binding via the increase in mant
fluorescence, because we observed a decrease in Trp fluorescence over time in
the absence of mant-GTP (data not shown). We assume this is because of thermal
denaturation of the purified protein in the fluorometer cuvette. Whatever the
cause, by monitoring the increase in mant fluorescence we would slightly
underestimate rather than overestimate the rate of GTP binding. The distance
limitations of RET require that mant-GTP is bound to SR
for energy
transfer to occur. Therefore this method provides a sensitive means to compare
the rate of GTP binding to SR
TM and
SR
C71D
TM. Reactions containing 300 nM
SR
and 500 nM mant-GTP were excited at 280 nm, and energy
transfer was monitored by measuring the increase in mant-GTP fluorescence
emission at 340 nm (Fig.
4b). Both SR
TM and
SR
C71D
TM bound mant-GTP, with SR
TM
following biphasic binding kinetics. The first mode is complete after 45 min;
the second mode is slower and takes an additional hour to complete.
SR
C71D
TM shows a single mode of GTP uptake that is
complete after 45 min. The initial rate of increase in mant fluorescence is
greater for SR
C71D
TM than for SR
TM,
indicating that the cysteine mutation permits mant-GTP more rapid access to
the GTP binding site in SR
.
SR contains only one Trp located five amino acids from the carboxyl
terminus. Excitation at 295 nm permits measurement of RET between this
tryptophan and mant-GTP. No change in the apparent kinetics of GTP binding to
SR
C71D
TM was observed
(Fig. 4c). However,
SR
TM now showed a single mode of GTP binding, that resembles the
second mode detected at 280-nm excitation in both slope and duration.
Therefore, the first mode arises from RET between one or more of the Tyr (and
Phe) residues scattered throughout SR
TM and mant-GTP, whereas the
second mode arises from RET between the carboxyl-terminal Trp residue and
mant-GTP.
Although fluorescence spectroscopy permits determination of binding
kinetics it did not permit us to determine what fraction of SR can bind
GTP. Therefore, a nitrocellulose filter binding assay was used to quantify the
amount of GTP that could bind SR
(Fig. 5). 100 pmols of
SR
TM (diamonds) or SR
C71D
TM
(squares) was incubated with a 10-fold molar excess of GTP, including
25% [3H]GTP, at 24 °C for the indicated times. To analyze GTP
binding the protein was bound to nitrocellulose filters and washed extensively
to remove unbound nucleotide. The nucleotide remaining on the filter,
representing the amount of solution GTP retained in a complex with SR
,
was quantified by scintillation counting. At 24 °C both proteins show that
same t
for nucleotide binding as calculated from
fluorescence data. After 2 h SR
TM bound a maximum of 6.6 pmols of
GTP, reflecting an occupancy of 6.6%. SR
C71D
TM bound
GTP at a faster rate than SR
TM, reaching a maximum of 3.5 pmols
of GTP bound after 1 h followed by a steady decline throughout the rest of the
experiment. A similar decline in binding was observed during RET experiments
(Fig. 4) and may reflect
structural instability of SR
C71D
TM during extended
incubation at 24 °C. These data reveal that <10% of SR
binds
exogenous GTP (de novo or by exchange). Therefore, 90% of SR
is
already tightly bound to nucleotide or in a conformation that is unable to
bind nucleotide. Both RET and filter binding experiments indicate that
SR
binds added GTP slowly, consistent with observations made in other
GTPases assayed in their nucleotide-bound states
(30,
35,
36).
The Kd of SR for GTP has been reported
to range from 1 µM for the purified, solubilized SR dimer
(12) to 20 nM for
the purified SR dimer reconstituted into liposomes
(18). To determine the
Kd of the 7% of SR
that can accept
exogenous GTP, purified SR
was cross-linked to
[
-32P]GTP in the presence of an increasing concentration of
cold competitor GTP (Fig. 6,
). Binding follows a characteristic sigmoidal curve with the inflection
point occurring at 2 µM, demonstrating that recombinant SR
and solubilized SR (12) have
similar affinities for GTP. An identical Kd was
calculated for SR
C71D
TM
(Fig. 6,
). Therefore
purified recombinant SR
TM binds GTP with a similar affinity as
native SR
after solubilization of microsomes, and mutation of the
cysteine in the G1 box does not affect the affinity of this protein for GTP.
Because of the short incubation period prior to cross-linking this
Kd measurement reflects the loose binding
conformation revealed by RET and not the majority of SR
that is already
bound to GTP.
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Because the majority of SR remains bound to GTP throughout
purification (Fig. 3) it is
likely that the intrinsic GTPase activity of SR
TM is negligible.
To experimentally verify that the SR
GTPase does not possess intrinsic
catalytic activity, SR
TM was incubated with
[
-32P]GTP, and hydrolysis was monitored by quantifying the
liberation of the terminal phosphate by thin layer chromatography
(Fig. 7). RNCs treated with
N-ethylmaleimide (NEM), identical to those used in previous attempts
to assay the influence of the ribosome on the SR
GTPase
(18), demonstrated that NEM
treatment is not sufficient to abolish GTPase activity associated with RNCs
(Fig. 7a,
NEM-RNC). Therefore, initial measurements were made in the
absence of ribosomes. After 4 h of incubation at 24 °C no significant GTP
hydrolysis was visually apparent above a control reaction lacking SR
(Fig. 7a,
GTP). To ensure that the assay was sensitive enough to measure a low
basal rate of GTP hydrolysis, the assay was repeated with varying
concentrations of GTP, and a Lineweaver-Burke plot was generated to estimate a
basal GTP hydrolysis rate (Fig.
7b). From the plot an estimated
Km of 4.0 µM and
kcat of 0.0005 min1 are
derived, reflecting a negligible rate of GTP hydrolysis for SR
TM.
Consistent with the negligible rate of GTPase activity obtained using the thin
layer chromatography assay, the efficiency of cross-linking
[
-32P]GTP and [
-32P]GTP to SR
were
identical (data not shown). Therefore SR
is unable to hydrolyze GTP,
suggesting the existence of a GAP.
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A candidate GAP for SR has been proposed recently
(18) to reside within the
ribosome. Because ribosomes are a significant source of GTPase activity, this
activity must be abolished to unambiguously assign GTPase activity arising
from SR
in reactions containing both SR
and ribosomes. The use of
alkylating reagents to modify ribosomal proteins has been shown to reduce, but
not abolish, the activity of certain ribosome-associated GTPases
(37) (see also
Fig. 7a). Therefore in
addition to 80 S ribosomes, isolated ribosomal subunits and RNCs were treated
with NEM in an attempt to decrease background ribosome-associated GTPase
activity enough to detect GTP hydrolysis arising from SR
. Treatment with
NEM had no effect on the GTPase activity of 80 S ribosomes or RNCs (see
Fig. 7a and
Supplemental Fig. 1), but the
GTPase activity of isolated 60 S subunits, already significantly decreased
compared with intact ribosomes, was abolished following treatment with NEM
(see Supplemental Fig. 1).
Incubation of SR
with NEM-treated 60 S subunits did not result in any
additional GTP hydrolysis above background levels (data not shown).
Although isolated 60 S ribosomal subunits did not stimulate the SR
GTPase, it is possible that ribosome-associated GAP activity requires an
intact 80 S ribosome. The GTPase activity of 80 S ribosomes precluded analysis
with exogenous nucleotide; therefore we chose to analyze hydrolysis of GTP
bound by SR
TM upon incubation with RNCs. If a protein within the
ribosome acts as an SR
GAP then incubation of SR
with ribosomes in
the absence of added GTP should result in the hydrolysis of SR
-bound GTP
to GDP. If the ribosome functions as a guanine nucleotide releasing factor
then incubation of SR
TM with ribosomes should lead to the release
of SR
TM bound GTP or GDP. We assessed the effect of adding
ribosomes to SR
TM bound to GTP by removing the ribosomes by
centrifugation at the end of the incubation and then assayed for nucleotide in
the supernatant and bound to SR
TM using the HPLC method described
above. Using this approach we were unable to detect any increase in hydrolysis
of GTP because of the addition of ribosomes (data not shown). To increase the
sensitivity of the assay and ensure that there were excess ribosomes present
in the reaction we examined hydrolysis of 32P-labeled GTP using
thin layer chromatography as described above. Because nucleotide exchange in
SR
TM is very inefficient (see
Fig. 5) we incubated
SR
TM with [
-32P]GTP and EDTA. In other low
molecular weight GTPases this incubation step allows rapid nucleotide
exchange. The exchange reaction was stopped by adding excess
Mg2+
(25,
26). By monitoring the degree
of exchange by nitrocellulose filter binding it was discovered that even in
the presence of nucleotide and EDTA only 6% of SR
TM could
exchange GTP for [
-32P]GTP (data not shown), in agreement
with the results obtained from time-dependent nucleotide exchange
(Fig. 5). Unbound nucleotide
was removed by repurifying SR
TM on CM-Sepharose,
SR
TM was incubated alone or in the presence of >2-fold molar
excess of 80 S ribosomes, and GTP hydrolysis over time was monitored by TLC.
As expected, we detected no intrinsic GTPase activity in SR
TM
alone. Moreover, the presence of ribosomes did not stimulate the GTPase
activity of SR
TM (data not shown).
In addition to proposing that a ribosomal component acts as an SR
GAP, Bacher et al.
(18) measured a decreased
affinity between SR
and guanine nucleotides in the presence of
ribosomes, suggesting that a ribosomal component also behaves as a guanine
nucleotide releasing factor. However, if SR
displays a lower affinity
for GTP when incubated with ribosomes, it is likely that some GTP would
dissociate from SR
during the incubation and become available for
hydrolysis by the ribosome. Because we did not observe any GTP hydrolysis we
conclude that the GTP remains tightly bound to SR
throughout the
incubation with ribosomes.
Chemical cross-linking experiments have yielded a specific cross-link
between SR and a protein within the 60 S ribosomal subunit, suggesting
that a physical association does occur
(11). We were unable to detect
a cross-link between SR
and a ribosomal protein using conditions that
result in cross-links between SR
TM molecules and that in previous
publications supported cross-linking between ribosomes and SR
/SR
(see Supplemental Fig. 2). This
raised the possibility that we do not detect an influence of the ribosome on
SR
, because the ribosome is unable to bind SR
in the absence of
SR
. To test this possibility SR
binding to 80 S ribosomes and
RNCs was assessed by sedimentation in sucrose density gradients.
SR
TM or SR
C71D
TM was incubated with
purified 80 S ribosomes or RNCs, and ribosome-bound SR
was separated
from unbound SR
by centrifugation on a 1040% sucrose gradient.
Fractions were collected and analyzed by Western blotting with an antibody
against SR
(Fig. 8). Both
SR
TM and SR
C71D
TM formed a stable complex
with both untranslating ribosomes and RNCs, as revealed by their comigration
in sucrose (Fig. 8,
b-e). Prolactin, which is not expected to
interact with ribosomes, remained at the top of the gradient
(Fig. 8f). These data
demonstrate that although the ribosome is unable to stimulate the SR
GTPase, a stable interaction between the two can still occur. It should be
noted that SR
is present in excess over ribosomes in these experiments,
so it is not possible to estimate the percentage of SR
that is able to
bind to ribosomes from this figure. By performing the experiment with
equimolar amounts of SR
TM and ribosomes, we determined that 22%
of SR
is recovered in the ribosome-containing fractions (data not
shown). Although it is not possible to estimate the amount of SR
that
can initially form a complex with ribosomes, the fact that 22% of SR
remains bound to ribosomes throughout a 16-h centrifugation step provides
evidence that the interaction between ribosomes and SR
is stable.
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DISCUSSION |
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Consistent with previous data, we have shown that recombinant SR
binds GTP with a Kd of
2 µM,
similar to detergent-solubilized SR
(12), but much higher than the
Kd of SR
-SR
dimers reconstituted
into lipid vesicles (18). It
must be noted that for all of these reports the
Kd measurement is relevant only to the small
fraction of SR
that is able to bind solution GTP during the assay. In
our experiments greater than 70% of the SR
TM was already bound to
GTP, and less than 10% of the SR
TM added to the reaction binds
GTP prior to cross-linking (Fig.
5). Furthermore, the short incubation time used in cross-linking
studies favors the loose binding conformation
(Fig. 4). The affinity for GTP
of the larger population of SR
that purifies bound to GTP is unknown,
but it is presumably much higher than 2 µM, because there is no
appreciable exchange with solution GTP during an 8-h incubation
(Fig. 5), and bound GTP is
removed during purification of the protein extremely slowly or not at all,
despite the absence of solution GTP in the purification buffers.
Previous attempts to measure the Kd of
SR (12) did not account
for the possibility that much of the SR
used in the assay may not be
able to accept exogenous GTP. It is likely that these
Kd measurements reflect the affinity of the same
small population of SR
measured here that can bind to (or exchange with)
exogenous GTP and do not reflect the affinity of the majority of SR
in
the assay. It is perhaps significant that 23% of SR
purified from
E. coli is bound to GDP. Our estimates of the fraction of SR
that binds GTP, 36%, are similar enough to 23% that we speculate
that the GDP bound form of SR
is responsible for the binding activity
that we measured. Bacher et al.
(18), by incorporating
purified SR into proteoliposomes, have measured a
Kd in close agreement with other Ras-type
GTPases. It is possible that lipid binding by SR
leads to a
conformational change that permits exchange of bound GTP with exogenous
GTP.
The GTP binding site in SRTM differs from other low molecular
weight GTPases in that it contains a cysteine at a position within the G1
GTPase consensus sequence that is highly conserved as either glycine or
aspartic acid in other family members of Ras-type GTPases
(Table I). Structural analysis
of Ras (39) and ARF-1
(40) does not provide insight
into the functional role of the amino acid at this position, but mutation of
this residue is invariably detrimental to the function of the protein
(16,
17,
41,
42).
The identity of the amino acid at this position in SR is somewhat
less conserved, suggesting that the side chain at this position is less
important for the function of the protein than it is for other GTPases
(Table II). In yeast, SR
contains a glutamine at this position that simultaneously binds SR
and
protrudes into the SR
GTP binding pocket
(13). SR
shows greater
sequence homology to Arf GTPases than to Ras
(12); therefore we mutated the
Cys to Asp in an attempt to convert SR
into an Arf-type GTPase. Compared
with wild type, SR
C71D
TM appears to bind GTP with
faster kinetics than SR
TM, suggesting that the conformation of
the protein has changed such that GTP has easier access to the binding pocket.
The cysteine normally at this position may contribute to the stability of the
protein, because at 24 °C the mutant protein exhibits a gradual loss of
nucleotide binding (see Figs. 4
and 5). Nucleotide preference
was not affected by this mutation, because fluorescence assays failed to
detect binding of mant-XTP to SR
C71D
TM (data not
shown). We were also unable to distinguish a difference in nucleotide affinity
between SR
TM and SR
C71D
TM for the small
fraction of protein that binds exogenous nucleotide. Unlike the Asn in yeast
SR
TM, the Cys in the canine protein is not likely to be involved
in binding of SR
TM to SR
, because the C71D mutation does
not appear to interfere with coimmunoprecipitation of SR
with
SR
C71D
TM (Fig.
1). Finally, we were unable to detect GTPase activity arising from
SR
C71D
TM (Fig.
8) (data not shown), but because we could not detect GTPase
activity from wild-type SR
TM the role of the cysteine in
catalysis remains uncertain.
The use of fluorescence to study GTP binding to SRTM revealed
a two-step process (Fig. 4).
The first step is rapid and was detected by monitoring energy transfer between
Tyr (and Phe) residues within SR
and a mant fluorophore incorporated
into GTP. The second step is slower and was detected by monitoring energy
transfer between a Trp residue located at the carboxyl terminus of SR
and the mant fluorophore. This second step occurs on the same time scale as
GTP binding monitored by a nitrocellulose filter binding assay, a technique
that captures tightly bound protein-nucleotide complexes
(Fig. 5). Taken together, these
data suggest a two-step model for GTP binding to SR
.
The first step involves GTP bound to SR in a loose conformation. The
filter binding assay does not detect these complexes as loosely bound GTP is
washed away. We detect these complexes by energy transfer between SR
and
mant-GTP. The time scale of this loose binding interaction is similar to GTP
binding by other Ras-type GTPases assayed in their GDP-bound state
(30,
35). The second step
represents a tight binding conformation, detected by both filter binding and
energy transfer between Trp and mant-GTP. Our data suggest that in
SR
TM the carboxyl terminus of SR
reorients such that energy
transfer occurs between Trp and mant. We propose that this conformational
change stabilizes the tight binding GTP-SR
TM complex. Comparison
of the data in Fig 4, b and
c suggests that the increase in RET between the Trp and
mant-GTP occurs subsequent to binding. The simplest explanation for the
increase in RET with the Trp is that SR
TM undergoes a
conformational change that moves the Trp closer to the GTP binding site.
Consistent with a role for the carboxyl terminus of SR
in stabilizing
the structure of the protein we have shown previously
(21) that the
carboxyl-terminal six amino acids (including the one Trp in SR
) are
required for folding of the protease-resistant core of SR
.
SRC71D
TM exhibits tight binding of GTP but does not
undergo the conformational change that stabilizes the complex, because the
rate of GTP binding is the same whether it is measured by RET or filter
binding. It may be that access to the GTP binding site is altered in
SR
C71D
TM but the mechanism of GTP binding is not
changed.
We have shown that the bulk (70%) of both SR
TM and
SR
C71D
TM are tightly bound to GTP. In contrast other
Ras-type GTPases are all purified in the GDP-bound state
(2830).
Even ARF-1 purifies GDP-bound, yet it exhibits no measurable GTPase activity
in vitro (29,
35). Ran purifies bound to
both GTP and GDP, reflecting the equilibrium between the two populations
within the cell (43). Thus,
SR
is the only Ras-type GTPase confirmed to purify predominantly in the
GTP-bound state. This result is not specific for isolated SR
, because
SR
-SR
complexes also purified in the GTP-bound state
(13).
The finding that SR defaults to the GTP-bound state whereas other
Ras-type GTPases default to the GDP-bound state makes some biological sense.
GTP-bound SR
binds to SR
more tightly than when SR
is
loaded with other nucleotides
(21). Because SR
is
required to anchor SR
to the ER membrane, and SR
is found almost
exclusively in the membrane fraction
(38), unlike other Ras-like
GTPases it makes sense to keep SR
bound to GTP.
It has been reported previously
(18) that ribosomes both
stimulate the SR GTPase and decrease the affinity of SR
for
nucleotides. Furthermore, an interaction between SR
and a 21-kDa
ribosomal protein has been detected, which may provide the basis for the
effect of the ribosome on nucleotide binding by SR
(11). However, in the absence
of SR
, we were unable to detect an influence of ribosomes or ribosomal
sub-units on the SR
GTPase. Attempts to detect a cross-link between a
ribosomal protein and SR
incubated with GTP or GDP were also
unsuccessful (see Supplemental Fig.
2). Because SR
can bind to ribosomes in the absence of
SR
these results suggest that SR
changes the quality of the
interaction between the ribosome and SR
, either by regulating the
structure of SR
to facilitate GTP hydrolysis or by directly contributing
residues that are required for GTP hydrolysis. This suggests an additional
role for SR
in translocation as an effector of SR
and is
consistent with structural data that demonstrate that SR
binds SR
predominantly through the SR
switch I region
(13).
Ribosomes are unlikely to interact with SR in the absence of
SR
in vivo. Therefore, an SR
-SR
-ribosome-nascent
chain-translocon complex may be required for SR
to hydrolyze GTP.
Hydrolysis of GTP may then lead to dissociation of SR
from SR
,
contributing to transfer of the RNC from SR to the translocon
(21).
We have shown previously that SR forms a tight physical association
with SR
, and this interaction is nucleotide-dependent and requires the
intact GTPase domain of SR
and is facilitated by the unique loop
sequence located between the G4 and G5 boxes
(20,
21). These characteristics are
consistent with SR
assuming the role of an SR
effector molecule.
Our finding that the ribosome does not stimulate the GTPase activity of
isolated SR
, together with previous data demonstrating GTPase activity
of SR
only in the context of an SR
-SR
-ribosome complex
(18), leads us to speculate
that SR
may regulate the GTPase activity of SR
. A detailed
structural analysis of SR
alone and in a complex with SR
will be
required to assess the extent that binding of SR
regulates SR
function.
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FOOTNOTES |
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The on-line version of this article (available at
http://www.jbc.org)
contains two supplemental figures.
Holds the Canada Research Chair in Membrane Biogenesis. To whom correspondence
should be addressed: Dept. of Biochemistry, McMaster University, 1200 Main St.
W., Hamilton, Ontario L8N 3Z5, Canada. Tel.: 905-525-9140 (ext. 22075); Fax:
905-522-9033; E-mail:
Andrewsd{at}fhs.mcmaster.ca.
1 The abbreviations used are: ER, endoplasmic reticulum; SR, signal
recognition particle receptor; GAP, GTPase activating protein; RET, resonance
energy transfer; RNC, ribosome-nascent chain; mant-GTP, 2' (or
3'-)-O-(N-methylanthraniloyl)-GTP; SRP, signal
recognition particle; XTP, xanthosine 5'-triphosphate; EK, enterokinase;
Ni-NTA, nickel-nitrilotriacetic acid; DTT, dithiothreitol; HPLC, high
performance liquid chromatography; NEM, N-ethylmaleimide.
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ACKNOWLEDGMENTS |
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REFERENCES |
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