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J. Biol. Chem., Vol. 278, Issue 26, 23686-23690, June 27, 2003
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-Subunit of the Protein-conducting Channel of the Endoplasmic Reticulum Functions as the Guanine Nucleotide Exchange Factor for the -Subunit of the Signal Recognition Particle Receptor*
From the Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021
Received for publication, April 29, 2003 , and in revised form, May 12, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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- and 
-subunits of
the SRP receptor (SR). SR is related to ADP-ribosylation factor
(ARF)-type GTPases, and the recently published crystal structure of
SR-GTP in complex with the binding domain of SR suggested that
SR, like all ARF-type GT-Pases, requires a guanine nucleotide exchange
factor (GEF) for function. Searching the sequence data base, we identified
significant sequence similarity between the Sec7 domain of ARF-GEFs and the
cytosolic domains of the -subunits of the two homologous heterotrimeric
protein-conducting channels in yeast. Using a fluorescence nucleotide exchange
assay, we show that the -subunits of the heterotrimeric
protein-conducting channels function as the GEFs for SR. Both the
cytosolic domain of Sec61 as well as the holo-Sec61, when part of
the isolated trimeric Sec61p complex, function as the GEF for SR,
whereas the same Sec61, when part of the heptameric complex that
facilitates posttranslational protein transport, is inactive as the GEF for
SR |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The GTPase domains of SRP54 and SR are very similar in their
structure and function. Structural studies of their prokaryotic homologues
have shown that they form their own family within the GTPase superfamily
(3,
4). They have low affinity for
nucleotide and are both stable in their empty states
(5,
6). SRP54 binds GTP when in
contact with the RNC (6). The
interaction between SRP54 and SR leads to the formation of a GTP
stabilized SRP-SR complex
(7). GTP hydrolysis dissociates
the SRP-SR complex (8),
whereby SRP54 and SR serve as mutual GTPase-activating proteins (GAP).
This reciprocally symmetric interaction is unique among known GTPases
(9).
SR is closely related to members of the ADP-ribosylation factor (ARF)
GTPase family (10,
11). ARFs are conserved in all
eukaryotes and are involved in the regulation of vesicle transport
(12). SR is present only
in eukaryotes and contains a transmembrane segment providing the membrane
anchor for SR (13).
However, a truncated protein representing the cytosolic GTPase domain without
the transmembrane domain is functional in protein targeting
(14). The function of the
GTPase domain of SR has long been a matter of conjecture. A recent
structural and biochemical study showed that SR effectively binds
SR only when bound to GTP and not when bound to GDP
(11), revealing that the GTP
cycle of SR controls the association and dissociation of the
heterodimeric SR.
The precise order of events that lead to the disassembly of the RNC-SRP-SR
complex and the transfer of the signal sequence to the PCC are unknown. The
PCC is composed of an oligomeric assembly of heterotrimeric integral membrane
proteins: Sec61, -, and -
. In yeast there are two
homologous -subunits (termed Sec61p and Ssh1p) and two homologous
-subunits (termed Sbh1p and Sbh2p). The two - and -subunits
form distinct trimeric complexes (termed the Sec61p and Ssh1p complex), each
with the shared -subunit (termed Sss1p)
(15–17).
In comparison to the Sec61p complex, the Ssh1p complex interacts with signal
sequences of stronger hydrophobicity
(18) and appears to be
involved exclusively in cotranslational protein transport. The trimeric Sec61p
complex can associate with four additional subunits Sec62p, Sec63p, Sec71p,
and Sec72p to form the so called heptameric complex
(19), which facilitates
posttranslational protein transport in yeast.
To identify a GEF for SR, we have searched the sequence data base for
homologues of the known GEFs for ARF related GTPases in yeast. We found that
the cytosolic domains of each of the -subunits of the PCCs, Sbh1p and
Sbh2p, showed homology to a motif that is common to ARF specific GEFs. We have
employed a fluorescence spectroscopy technique to directly monitor the
nucleotide exchange of SR and have shown that the cytosolic domains of
recombinant Sbh1p and Sbh2p indeed function as the GEFs for SR. GEF
activity was retained when Sbh1p was part of the trimeric Sec61p complex. In
contrast, GEF activity was not expressed when Sbh1p was part of the heptameric
complex that functions in posttranslational translocation. We propose a model
in which Sbh1p and Sbh2p regulate the reassociation of the SR subunits,
thereby coupling targeting with translocation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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with MantGDP—The
cytosolic GTPase domain of SR from Saccharomyces cerevisiae
(residues 31–244) (11)
was expressed in BL21(DE3) cells (Stratagene). SR31–244 was
purified by cobalt affinity chromatography (TALON Metal Affinity Resin,
Clontech) from two liters of cell culture and eluted in 15 ml of buffer A (20
mM Tris·HCl, pH 8.0, 250 mM NaCl, 5 mM
-mercaptoethanol) containing 150 mM imidazole. To remove
endogenous nucleotide bound to SR31–244, EDTA (0.5 mM)
was added to the eluate, followed by an incubation at 4 °C for 30 min. The
eluate was then subjected to size exclusion chromatography in the presence of
0.5 mM EDTA in buffer B (20 mM Hepes, pH 7.5, 250
mM NaCl, 1 mM DTT) (Superdex-75, Amersham Biosciences).
Fractions of a single A280 peak, representing
SR31–244, were pooled and stored at –80 °C.
A 10 µM solution of SR31–244 was incubated for 30
min at 4 °C with 200 µM of
3'-(N-methylanthraniloyl)-2'-deoxyguanosine-5'-diphosphate
(mantGDP) in buffer C (20 mM Hepes, pH 7.5, 350 mM KOAc,
1 mM DTT, and 5 mM Mg(OAc)2). The excess of
unbound mantGDP was removed by dialysis against buffer C at 4 °C for 4
h.
Purification of the Heptameric and the Sec61p Complex—The heptameric complex was immunopurified from yeast strain Sec63prA, in which the gene coding for Sec63p was tagged by in-frame integration of a DNA fragment encoding for a Factor X protease site and the immunoglobulin G (IgG)-binding domains of protein A, yielding a fusion protein of Sec63p-FactorX site-protein A (20). Cells from a 36-liter culture were lysed in lysis buffer (20 mM Hepes, pH 7.5, 100 mM KOAc, 5 mM Mg(OAc)2, 1 mM DTT, 0.5 mM PMSF, 125 mM sucrose, and protease inhibitors (complete protease inhibitor mix, Roche Molecular Biochemicals)) at 13,000 p.s.i. using a French press. The cell lysate was cleared by low speed centrifugation at 6,000 x g for 15 min at 4 °C and then centrifuged at 100,000 x g for 38 min at 4 °C to obtain a crude membrane pellet. Aliquots of the crude membrane pellet were stored at –80 °C. A 10th part of the crude membrane pellet (5 ml) was solubilized by resuspending it in 46 ml of solubilization buffer (20 mM Hepes, pH 7.5, 400 mM sucrose, 750 mM KOAc, 2.5 mM Mg(OAc)2, 0.5 mM EDTA, 3% digitonin (Calbiochem), 5 mM DTT, and 0.5 mM PMSF) and incubation on ice for 30 min. Insoluble material was pelleted by centrifugation at 100,000 x g for 30 min at 4 °C. The supernatant was diluted 1:1 with dilution buffer (20 mM Hepes, pH 7.5, 400 mM sucrose, 2.5 mM, Mg(OAc)2) and incubated over night at 4 °C with 1 ml IgG-Sepharose (Cappel, Durham, NC). The resin was washed using washing buffer WLX (20 mM Hepes, pH 7.5, 100 mM KOAc, 3 mM Mg(OAc)2, 10% glycerol, 1 mM DTT) containing 0.5% digitonin and 5 mg/ml asolectin and the heptameric complex eluted by incubating with 5 µg/ml Factor X protease in 1 ml of WLX buffer for 5 h at 25 °C. To exchange detergents, pooled fractions containing the heptameric complex were incubated with Q-Sepharose (Amersham Biosciences), equilibrated and eluted with buffer WLX containing 0.3% DeoxyBigCHAP (Calbiochem).
The trimeric Sec61p complex was essentially purified as described previously (20). Briefly, the immunomobilized heptameric complex (see above) was incubated with buffer WLX containing 1% Triton X-100, thereby dissociating the trimeric Sec61p complex from the heptameric complex. To exchange detergent, the trimeric Sec61p complex was then bound to SP-Sepharose (Amersham Biosciences), equilibrated and eluted with buffer WLX containing 0.3% DeoxyBigCHAP.
Purification of Sbh1p and Sbh2p—DNA coding for the
NH2-terminal cytosolic domain of Sbh1p (residues 1–53) or
Sbh2p (residues 1– 61) was subcloned into pGEX4-T2 (Amersham
Biosciences) and pGEX6-P1 (Amersham Biosciences), respectively. Sbh1p
C
was expressed in BL21(DE3) cells (Stratagene) and Sbh2pC was expressed
in BL21(DE3)-RIL cells (Stratagene). The GST-tagged proteins were isolated
from cleared Escherichia coli lysates by affinity chromatography
using glutathione-Sepharose (Amersham Biosciences).
SR Nucleotide Exchange Assay—Steady-state
fluorescence intensity was measured using a SPEX Fluorolog FL3-11
photon-counting spectrofluorimeter with a single grating excitation
monochromator and a 450-watt xenon lamp. Samples were exited at 352 nm (1 nm
bandpass) and emission was detected at 440 nm (5-nm bandpass). All spectral
measurements were done at 25 °C in buffer J (20 mM Hepes, pH
7.5, 350 mM KOAc, 5 mM Mg(OAc)2, 10%
glycerol, 1 mM DTT, 100 µM GDP, and 0.3%
DeoxyBigCHAP) using a 4 x 4-mm quartz cuvette. The release of mantGDP
from SR31–244 was measured in real time as a decrease in
fluorescence.
Purification of Ribosomes and Ribosome Nascent Chain Complexes— Ribosomes and ribosome nascent chain complexes were purified as described previously (2).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ARF into account, a similarity between the
respective GEFs is also conceivable. Searching the sequence data base, we
could identify significant sequence similarity between the cytosolic domains
of the two -subunits of the PCCs in yeast and the Sec7 domain
(Fig. 1). The sequence
similarity involves the region of Sec7 that is crucial for GEF function,
namely the F-G loop and helix H
(21). Based on these findings,
we tested whether Sbh1p and Sbh2p are able to promote the exchange of
nucleotide for SR.
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Sbh1p and Sbh2p Function as the GEFs for SR—A
fluorescence spectroscopic assay was used, which allows one to monitor the
nucleotide exchange on a G-protein in real-time and at high resolution. It had
been shown that the fluorescent signal of mantGDP is dependent on the
environment of the probe and hence on the amount of mantGDP bound by a
G-protein (22). Recombinant
yeast SR31–244 was purified from E. coli and endogenously
bound nucleotide removed by size exclusion chromatography in the presence of
EDTA. The removal of nucleotide was confirmed by high performance liquid
chromatography analysis and determined to be complete (data not shown).
SR31–244 was reloaded by incubation with an excess of mant-GDP and
unincorporated mantGDP was removed by dialysis. The reloading of
SR31–244 with mantGDP resulted in a substantial increase of
emitted fluorescence compared with mant-GDP alone. The
SR31–244-dependent increase averaged to 20%, indicating that
mantGDP was successfully bound by SR31–244
(Fig. 2A).
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The SBH1 gene in yeast codes for an 8.7-kDa protein with a COOH-terminal
transmembrane domain. Its homologue Sbh2p is slightly larger with a size of
9.6 kDa. To test whether Sbh1p or Sbh2p are directly involved in the
nucleotide exchange of SR, we purified the recombinant
NH2-terminal cytosolic domains of Sbh1p (Sbh1pC) and Sbh2p
(Sbh2pC) by GST affinity chromatography
(Fig. 2D, as
indicated). Equimolar amounts of mantGDP-SR31–244,
GST-Sbh1pC, or GST-Sbh2pC in the presence of an excess of GDP
were incubated at 25 °C. The reactions were excited at 352 nm and the
emission recorded at 441 nm. When incubating mock buffer with
mantGDP-SR31–244, no decrease of fluorescence could be detected
(Fig. 2B). This result
indicates that SR31–244 binds nucleotide tightly and does not
exchange with the solution by itself. When using GST alone in the assay, no
decrease in fluorescence could be detected either (data not shown). In the
presence of GST-Sbh1pC or GST-Sbh2pC, a substantial decrease of
fluorescence could be measured resulting from the exchange of nucleotide. The
observed decrease of fluorescence averaged 12% after 15 min
(Fig. 2B) and reached
a plateau after a decrease of 14% (data not shown). The obtained data were
best fitted as a sum of two exponential decays. When a 10-fold excess of
either GST-Sbh1pC or GST-Sbh2pC over SR31–244 was
used in the assay, the decrease of fluorescence averaged 50%, indicating that
GST-Sbh1pC and GST-Sbh2pC do exchange the nucleotide of SR
stoichiometrically rather than catalytically (data not shown).
Sbh1p Functions as the GEF for SR in the Trimeric Sec61p
but Not in the Heptameric Complex—Sbh1p is assembled into two
different PCCs in yeast. It is not only part of the trimeric Sec61p complex,
involved in cotranslational protein transport, but also part of the heptameric
complex that is exclusively involved in posttranslational protein transport.
To test whether the GEF activity for SR is dependent on the assembly
state of Sbh1p we purified the trimeric Sec61p and the heptameric complex from
yeast (Fig. 2D, as
indicated) and tested both detergent-solubilized complexes in the nucleotide
exchange assay.
We incubated equimolar amounts of mantGDP-SR31–244 with
trimeric Sec61p complex or heptameric complex in the presence of an excess of
GDP at 25 °C. When mantGDP-SR31–244 was incubated with
purified trimeric Sec61p complex, mantGDP was efficiently exchanged with GDP,
resulting in a decrease of fluorescence that averaged to 17% after 15 min
(Fig. 2C) and reached
a plateau after a decrease of 20% (data not shown). The obtained data were
best fitted as a sum of two exponential decays. When using equimolar amounts
of mantGDP-SR31–244 and purified heptameric complex, no decrease
in fluorescence could be detected (Fig.
2C).
The ribosome had been reported to reduce the affinity of SR for
nucleotide (23) and therefore
was proposed to function as the GEF for SR
(24). To test whether the
ribosome is able to promote the exchange of nucleotide for SR, we used
purified translating and non-translating ribosomes in the GTPase exchange
assay. In our system, neither translating nor non-translating ribosomes were
able to exchange the nucleotide of SR (data not shown).
We conclude that Sbh1p and Sbh2p are the GEFs for SR and are able to
promote the exchange of nucleotide without the other subunits of the trimeric
Sec61p complex. Furthermore, Sbh1p is able to promote the nucleotide exchange
for SR when assembled into the trimeric Sec61p complex but not when
assembled into the heptameric complex.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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requires a GAP and GEF to complete its GTP switch cycle
(11). Without a GEF, the
nucleotide exchange in most GTPases occurs very slowly under physiological
conditions (12). In agreement
with this, we could not detect any intrinsic nucleotide exchange activity of
purified SR, which indicates that SR relies on an extrinsic
GEF.
Searching the sequence data base, we have identified a sequence similarity
between the -subunits of the two homologous trimeric PCCs in yeast and
the conserved Sec7 domain that is present in all ARF-GEFs. The residues in
contact with ARF1 in the ARF1-Sec7 complex form a binding groove made up of
two regions: the -helix H and the loop connecting the preceding helices
F and G of the all--helical Sec7 domain
(21), forming a functional
module. This module is not a folded domain, but is rather held in shape by the
remainder of the Sec7 domain, which forms the surrounding architectural
scaffold. Therefore it appears that the SR binding groove of Sbh1p/Sbh2p
is presented in a different structural context. Sbh1p and Sbh2p are not
related to the Sec7 domain except for the functional module. The cytosolic
domains of Sbh1p and Sbh2p consist of only about 60 residues and thus
represent the smallest functional GEFs to date. We note that not all residues
that are important for Sec7 function are conserved in Sbh1p/Sbh2p. Therefore
the guanine nucleotide exchange of SR will also differ from ARF GEFs in
some detail.
Using a fluorescence nucleotide exchange assay, we show that recombinant
proteins comprising the cytosolic domains of Sbh1p and Sbh2p indeed function
as the GEFs for SR. Despite the small size of their cytosolic domains,
Sbh1p and Sbh2p are able to efficiently promote the exchange of nucleotide for
SR. We find that Sbh1p is also functional as the GEF for SR when
assembled into the detergent-solubilized trimeric Sec61p complex isolated from
yeast. A direct interaction between the trimeric Sec61p complex and SR
is supported by the fact that a protein interaction screen has found the
trimeric Sec61p complex and its homologue, the Ssh1p complex to be in the
proximity of SR (18).
The calculated reaction rates at which the trimeric Sec61p complex, Sbh1p, and
Sbh2p promoted nucleotide exchange are very similar, which indicates that both
-subunits promote the nucleotide exchange equally well, independent of
their assembly state with the other subunits of the trimeric complexes.
Interestingly, however, when Sbh1p was assembled into the
detergent-solubilized heptameric complex isolated from yeast, it did not
express its GEF activity. This suggests, that the cytosolic domain of Sbh1p in
this complex might not be accessible to SR.
The -subunit of the SR is an eukaryotic feature; the SR
homologue in bacteria directly associates with the membrane
(1). Interestingly, there is no
bacterial homologue for Sbh1p. It is tempting to speculate that in higher
organisms, the SR subunit evolved in concert with its GEF, not merely to
provide a membrane anchor for SR but also to provide an additional
regulatory step to the translocation process; the interaction between these
two proteins in the eukaryotic ER membrane, one a component of the targeting
machinery (SR), the other a component of the trimeric PCCs, suggests that
these proteins might link the two processes in a controllable fashion.
The association of the SR subunits is controlled by the nucleotide bound
state of SR (11). With
SR in its GDP bound form, the SR subunits dissociate. The
-subunits of the trimeric PCCs, acting as the GEFs for SR, reload
SR with GTP and therefore control the reassociation of the SR subunits.
It is conceivable that the -subunits of the occupied PCC are
inaccessible to SR. This would prevent the nucleotide exchange and the
heterodimerization with SR. As a result the targeting machinery could
not be linked to the occupied PCC. Sbh1p can be cross-linked to Spc25p, a
subunit of the signal peptidase complex
(25). Inaccessibility of the
-subunits of the PCCs may, therefore, not only be caused by the RNC
complex binding to the PCCs, but also by the lateral recruitment within the
plane of the ER membrane of other integral ER membrane proteins, such as the
signal peptidase complex, the oligosaccharyltransferase or the additional
subunits recruited to form the heptameric complex which is involved in
posttranslational transport.
It had been reported that the ribosome decreases the affinity of SR
for nucleotide (23). From
these results it had been concluded that the ribosome might stabilize an empty
state of SR and therefore could function as its GEF. However, using
either purified non-translating or translating ribosomes in our assay had no
effect on the nucleotide exchange of SR.
Our data show a specific function for the -subunits of the two
homologous PCCs and allows for a refined model of the cotranslational
targeting process that had previously been suggested
(11)
(Fig. 3). In a first step, the
SRP recognizes the signal sequence and binds to the RNC complex. The
interaction between the ribosome and the SRP results in the binding of GTP by
SRP54 (6). Sbh1p assembled into
the trimeric Sec61p complex functions as the GEF for SR, loading it with
GTP. This nucleotide exchange reaction triggers the assembly of the SR
subunits in the proximity of the trimeric Sec61p complex. Only with the SR in
its assembled state, can the RNC-SRP complex be targeted to the ER membrane
where SRP54 and SR form a GTP stabilized complex. Next, the trimeric
Sec61p complex replaces the SRP at the exit site of the ribosome, the signal
sequence is transferred to the trimeric Sec61p complex and translocation
starts. GTP hydrolysis of SR would lead to the dissociation of the SR
subunits (11). In a last step,
SRP54 and SR act as mutual GAPs, thereby resolving the SRP-SR
interaction.
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The precise timing and coordination of the events during the signal
sequence transfer still remain poorly understood. Signal sequence transfer to
the trimeric Sec61p complex requires GTP binding by SRP54, SR and
SR but not necessarily GTP hydrolysis
(26). The events triggering
the GTP hydrolysis of the three GTPases and the release of the signal sequence
remain to be elucidated. Further biochemical characterization will be
necessary to completely understand the GTPase cycle of SR.
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FOOTNOTES |
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To whom correspondence should be addressed: Rockefeller University, HHMI, 1230
York Ave., Box 168, New York, NY 10021. Tel.: 212-327-8096; Fax: 212-327-7880;
E-mail:
helmerj{at}blobelsgi.rockefeller.edu.
1 The abbreviations used are: SRP, signal recognition particle; ER,
endoplasmic reticulum; GAP, GTPase-activating protein; ARF, ADP-ribosylation
factor; GEF, guanine nucleotide exchange factor; mant-GDP,
3'-(N-methylanthraniloyl)-2'-deoxyguanosine-5'-diphosphate;
PCC, protein-conducting channel; RNC, ribosome nascent chain complex; SR, SRP
receptor; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; GST,
glutathione S-transferase.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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