Correspondence to: Bernhard Dobberstein, Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Postfach 106249, 69052 Heidelberg, Germany. Tel:(6221) 546825 Fax:(6221) 545892 E-mail:dobberstein{at}zmbh.uni-heidelberg.de.
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Abstract |
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Protein targeting to the membrane of the ER is regulated by three GTPases, the 54-kD subunit of the signal recognition particle (SRP) and the - and ß-subunit of the SRP receptor (SR). Here, we report on the GTPase cycle of the ß-subunits of the SR (SRß). We found that SRß binds GTP with high affinity and interacts with ribosomes in the GTP-bound state. Subsequently, the ribosome increases the GTPase activity of SRß and thus functions as a GTPase activating protein for SRß. Furthermore, the interaction between SRß and the ribosome leads to a reduction in the affinity of SRß for guanine nucleotides. We propose that SRß regulates the interaction of SR with the ribosome and thereby allows SR
to scan membrane-bound ribosomes for the presence of SRP. Interaction between SRP and SR
then leads to release of the signal sequence from SRP and insertion into the translocon. GTP hydrolysis then results in dissociation of SR from the ribosome, and SRP from the SR.
Key Words: signal recognition particle receptor, GTPase, ribosome, translocation, endoplasmic reticulum
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Introduction |
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TARGETING of nascent secretory and membrane proteins to the membrane of the ER involves the interaction between the signal recognition particle (SRP)1 (
The targeting of nascent proteins to the ER membrane is regulated by three GTPases, SRP54, the 70-kD -subunit (SR
), and the 30-kD ß-subunit of SR (SRß;
Initial work showed that the SR-dependent release of the signal sequence from SRP requires GTP ( contributes to the stabilization of the SRPSR complex (
are in their GTP-bound form (
leads to the dissociation of SRP from its receptor (
(FtsY) suggest that these proteins function as GAPs for each other (
Little is known about the function of SRß and the regulation of its GTPase cycle. The ß-subunit is a type I integral membrane protein associated with the membrane by an NH2-terminal transmembrane segment (, and thereby anchors it to the membrane. The SR
can be released from the ß-subunit by carbonate extraction at pH 12.5 (
is released into the cytosol. Work with the yeast homologue of SRß has shown that the interaction between the two SR subunits is important for their function. Furthermore, it was found that a soluble form of SRß is also functional (
To investigate the GTPase cycle of the SRß, we analyzed GTP binding and hydrolysis in the presence of RNCs or SRP and liposomes containing SR, Sec61p complex, or translocating chain-associating membrane (TRAM) protein. Our results suggest that the ribosome contacts SRß in its GTP-bound state, stimulates GTP hydrolysis of SRß, and leads to a release of SRß-bound GDP.
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Materials and Methods |
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Materials
General chemicals were from Merck or Sigma Chemical Co. [32P]GTP (3,000 Ci/mmol), [35S]methionine, and the ECL system were purchased from Nycomed Amersham, Inc.
Purification and Reconstitution of SR, TRAM Protein, and Sec61p Complex
SR was purified by immunoaffinity chromatography (, coupled to keyhole limpet haemocyanin with sulphosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulpho-SMCC; Pierce Chemical Co.). The antibodies were affinity-purified against the immobilized peptide (Sulpholink gel; Pierce Chemical Co.) and then immobilized on protein ASepharose (Pharmacia Biotech, Inc.) with dimethylsubermidate. The affinity column was then used to purify SR from a digitonin extract of dog pancreas rough microsomes, essentially as described by
Sec61p complex was purified from a ribosome-associated membrane protein fraction by ion-exchange chromatography according to
Detergent exchange of translocon components from digitonin to deoxyBigCHAP, followed by reconstitution into proteoliposomes was performed as described (
Purification of SRP and RNCs
SRP was purified from a high salt extract of canine rough microsomes by gel filtration (Sephadex G-150), followed by ion-exchange chromatography (DEAESepharose) according to
RNC complexes bearing preprolactin 86mer nascent chains (PPL86) were synthesized in the wheat germ lysate translation system (
RNCs were finally resuspended in HMDC buffer (HMC with 1 mM DTT) and 150 mM KOAc at a concentration of 56 OD260 units/ml.
GTP Cross-linking Assay
SR, purified and reconstituted into liposomes (SR liposomes; [32P]GTP (3,000 Ci/mmol) or concentrations as indicated in 50 mM Tris-OAc, pH 7.8, 150 mM KOAc, 2 mM DTT, 5 mM Mg(OAc)2, 2 mM cycloheximide. After incubation for 20 min on ice and 5 min at 25°C, the 10 µl reactions were transferred onto a silanized glass plate precooled on ice-cold metal blocks and irradiated with UV light at 4,000 W/cm2 in a stratalinkerTM for 5 min to cross-link the radiolabeled GTP to the proteins (
GTP Hydrolysis Assay
SR, purified and reconstituted into liposomes ([32P]GTP (3,000 Ci/mmol) in 50 mM Tris-OAc, pH 7.8, 150 mM KOAc, 2 mM DTT, 5 mM Mg(OAc)2, and 2 mM cycloheximide at 25°C for the indicated time points.
Aliquots of the samples were spotted onto polyethyleneimine cellulose thin-layer plates; [32P]GDP was resolved from
[32P]GTP using 0.75 M KH2PO4, pH 3.3, as solvent. Radioactive TLC spots were quantitated using a PhosphorImager. The percentage of GTP hydrolysis was calculated from the amount of
[32P]GDP divided by the sum of the amounts of
[32P]GTP and
[32P]GDP.
Floatation Assay
Liposomes lacking or containing 50 nM SR or trypsinized SR were incubated in 50 mM Tris-OAc, pH 7.8, 150 mM KOAc, 2 mM DTT, 5 mM Mg(OAc)2, 2 mM cycloheximide with purified RNCs (2,8 OD260/ml) containing [35S]methionine-labeled PPL86 and 0.5 mM GMPPNP or GDP in the presence or absence of 50 nM SRP. Wheat germ cytosol (1.8 µl), which was previously depleted of ribosomes by pelleting the ribosomes at 400,000 g for 20 min, was subsequently added to 5 µl reactions to block unspecific binding of ribosomes to liposomes lacking SR. After 30 min incubation at 25°C, 60 µl of ice-cold sucrose buffer containing 2.2 M sucrose, 10 mM Tris-OAc, pH 7.8, 500 mM KOAc, 2 mM DTT, and 5 mM Mg(OAC)2 was added, thoroughly mixed, and layered under a 100 µl 1.8 M sucrose cushion containing CR buffer (50 mM Tris-OAc, pH 7.8, 500 mM KOAc, 2 mM DTT, 5 mM Mg(OAc)2, 2 mM cycloheximide, 0.5 mM guanine nucleotides) in a TLA 100 tube that was preincubated with a 10 mg/ml BSA/PBS solution. A 0.25 M sucrose cushion in CR buffer (40 µl) was laid over the 1.8 M sucrose cushion. After centrifugation at 400,000 g for 60 min, the amount of labeled PPL86 in the top and bottom fractions was determined by scintillation counting. The amount of labeled PPL86 that was recovered in the top fraction in the absence of liposomes was taken as background and was subtracted.
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Results |
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GTP Binding to SRß
To test GTP binding to SR subunits, we used UV light-mediated cross-linking of [32P]GTP to purified SR reconstituted into liposomes (SR liposomes). This approach allows analysis of GTP binding to each SR subunit (
[32P]GTP cross-linked to SR liposomes is revealed after SDS-PAGE followed by PhosphorImaging, and shows labeling of both SR
(70 kD) and SRß (30 kD). An unidentified protein of ~50 kD (Figure 1 A) was also found in various amounts, as has been reported previously (
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To determine the apparent affinity of SR for GTP, we added increasing concentrations of unlabeled GTP to the cross-linking reactions with
[32P]GTP. Proteins were analyzed by SDS-PAGE, the amount of label in SR
was quantified after PhosphorImaging, and was plotted against the concentration of added GTP. From these data, an apparent Kd of 14 µM was calculated (Figure 1 B). To determine the apparent affinity of SRß for GTP, we used increasing amounts of
[32P]GTP in the cross-linking reactions. The amount of labeled SRß was plotted against the concentration of
[32P]GTP (Figure 1 C). We determined an apparent Kd of 20 nM GTP for SRß. Thus, the affinity of SRß for GTP is ~700-fold higher than the affinity of SR
for GTP.
To test whether translocon components affect [32P]GTP cross-linking to the SR subunits, we used SR liposomes containing, in addition, the Sec61p complex and the TRAM protein. Figure 2 shows the purified proteins analyzed by SDS-PAGE and silver staining, and the proteins cross-linked to
[32P]GTP after SDS-PAGE and PhosphorImaging. As can be seen in Figure 2, cross-linking of
[32P]GTP to SR
and SRß is not changed by the inclusion of SR/Sec61p/TRAM liposomes to the assay (Figure 1 A and 2). Furthermore, we found that the Kd of SRß and SR
for GTP remained unchanged in the presence of SR/Sec61p/TRAM liposomes, as compared with SR liposomes (data not shown). Thus, we conclude that neither the TRAM protein, nor the Sec61p, influence
[32P]GTP cross-linking to the SR
or SRß.
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To test whether a component of the targeting complex affects GTP binding to SR, we added purified SRP and/or purified RNCs bearing PPL nascent chains of 86 amino acids to SR liposomes. As shown in Figure 3 A (lanes 1 and 2), the presence of SRP did not affect [32P]GTP cross-linking to either SR
or SRß. When RNCs were added,
[32P]GTP cross-linking to SR
remained the same, however,
[32P]GTP cross-linking to SRß was strongly reduced (Figure 3 A, lane 3). This indicates that the RNC interacts with SR and selectively reduces
[32P]GTP cross-linking to SRß. Reduction in
[32P]GTP cross-linking to SRß was also seen when SRP was added in addition to RNCs (Figure 3 A, lane 4). In this case, cross-linking of
[32P]GTP to SRP54 was found to be increased in the presence of RNC, as shown previously (Figure 3 A, compare lanes 2 and 4;
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The apparent affinity of SRß for GTP in the presence of RNCs was determined as described. We found that the apparent Kd was 1 µM, ~50-fold higher than that observed in the absence of RNCs (Figure 3 B). Cross-linking of [32P]GDP to SRß was also found to be reduced by the interaction with RNC (data not shown). This indicates that GTP, as well as GDP, binding to SRß is decreased by RNC.
We next asked whether the decrease in [32P]GTP cross-linking to SRß is caused by a direct interaction between RNCs and SRß, or if it requires the presence of both SR subunits. To test the latter possibility, we removed SR
by mild trypsin digestion (
was largely removed by treatment with 2 ng/ml trypsin and high salt, whereas SRß resisted proteolysis and remained bound to the membranes. Liposomes containing the ß-subunit of SR (SR
liposomes) were tested in the
[32P]GTP cross-linking assay. Strong
[32P]GTP cross-linking to SRß was seen, while
[32P]GTP cross-linking to SR
was greatly reduced (Figure 4 B, lane 1). The presence of SRP did not affect
[32P]GTP cross-linking to SRß (Figure 4 A, lane 2). When SR
liposomes were combined with RNCs,
[32P]GTP cross-linking to SRß was reduced similarly, as was seen with SR liposomes (Figure 4 B, lanes 3). SRP had no effect on the RNC-mediated reduction of
[32P]GTP cross-linking to SRß (Figure 4 B, lane 4). This indicates that RNCs directly interact with SRß, resulting in reduced
[32P]GTP cross-linking.
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GTP Hydrolysis by SR and SRß
We next investigated the effect of the RNCSR interaction on GTP hydrolysis. SR liposomes were incubated with RNC and/or SRP and GTP hydrolysis determined by chromatographic analysis of [32P]GDP generated in the assay. No significant GTP hydrolysis was observed with SR liposomes or RNC alone (Figure 5 A). However, when SR liposomes were combined with RNCs an increase of GTP hydrolysis was observed (Figure 5 A). This confirms that RNC interacts with SR and indicates that RNC stimulates GTP hydrolysis by SR. As shown previously, a large additional stimulation of GTP hydrolysis is observed when SRP is also added (
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To investigate the RNC-stimulated GTP hydrolysis of SR in more detail, we used increasing concentrations of RNCs in the assay. When SR liposomes were tested alone, or in the presence of SRP, we found that the amount of GTP hydrolyzed at a given time point was saturable (Figure 5 B). This indicates a specific interaction between RNC and SR.
To identify the subunit of the SR that hydrolyzes GTP in the presence of RNC, we used the SR liposomes containing only SRß. SR
liposomes alone, or combined with SRP, showed only background levels of GTP hydrolysis as observed with SR liposomes (Figure 5 C, lanes 1, 2, 5, and 6). When SR
liposomes were combined with RNC, the stimulation of GTP hydrolysis was similar to that observed with SR liposomes (Figure 5 C, lanes 3 and 7), suggesting that the RNC directly interacts with SRß and stimulates its GTP hydrolysis. Addition of SRP and RNC to SR
liposomes did not significantly enhance GTP hydrolysis above the level seen with RNC alone (Figure 5 C, lanes 3 and 4). A significant further stimulation of GTP hydrolysis is, as expected, observed with SR liposomes in the presence of RNC and SRP (Figure 5 C, lanes 7 and 8). This also has been observed previously, and reflects the reciprocal GTPase stimulation of SRP54 and SR
(
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GTP-dependent Interaction of RNC with SR and SR Liposomes
The observation that RNC reduces [32P]GTP cross-linking to SRß and stimulates GTP hydrolysis by SRß indicates that the RNC contacts SRß. To test this directly, we allowed interaction of RNC to SR liposomes and then floated SR liposomes with bound RNC to the top of a sucrose gradient to separate them from unbound RNCs. To test for a GTP dependence of this binding, we performed the assays in the presence of either GDP or the nonhydrolyzable GTP analogue GMPPNP. SR liposomes were incubated with purified RNCs bearing 35S-labeled PPL86. To reduce unspecific binding to the lipids, we included wheat germ cytosol from which endogenous ribosomes had been removed. SR liposomes were then floated and the amount of nascent chains (35S-labeled PPL86) associated with the floated SR liposomes and in the pellet was determined. 35S-labeled PPL86 nascent chains were not found associated with liposomes lacking SR (Figure 6, lane 1). In the presence of GDP, only a small amount of 35S-labeled PPL86 nascent chains were found associated with SR-liposomes (Figure 6, lane 2). In contrast, with GMPPNP, a significantly increased amount of nascent chains was recovered with the floated SR liposomes (Figure 6, lane 3). This suggests that RNCs bind to the SR liposomes in a GTP-dependent manner. To test the effect of SRP on RNC interaction with SR liposomes, we included SRP in the assay system. We found that, even in the presence of GDP, a further increase in RNC binding to SR liposomes compared with the absence of SRP (Figure 6, lanes 4 and 2). But, in the presence of GMPPNP, a substantially higher amount of RNCs was found associated with SR liposomes (Figure 6, lane 5). Taken together, this suggests two GTP-dependent interactions, one between the ribosome and SR and the other between SRP and SR.
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To test whether the RNC binds to SRß in the absence of SR, we used SR
liposomes in the floatation assay. As was seen with SR liposomes, a significantly higher amount of RNC floated with SR
liposomes in the presence of GMPPNP, as compared with GDP (Figure 6, lanes 6 and 7). These data suggest that the RNC directly interacts with SRß in a GTP-dependent manner. The addition of SRP in the assay led to an increased binding of RNC to SR
liposomes in the presence of GDP, as was seen with SR liposomes. This might point to a GTP-independent interaction between SRP and SRß. In the presence of GMPPNP, a further increase in binding was observed, but much less than observed with SR liposomes (Figure 6, lanes 5 and 9). This indicates that the binding of SRP to SR
is drastically reduced, whereas the binding between SRß and RNC is not affected (Figure 6, lanes 2, 3, 6, and 7).
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Discussion |
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The functions of GTPases are regulated by guaninenucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which mediate GTP binding and stimulation of GTP hydrolysis, respectively. Here, we show that the ribosome interacts with SRß in its GTP-bound state, functions as a GAP for SRß, and reduces the affinity of SRß for guanine nucleotides. Previously, it has been shown that the ribosome functions as a GEF for SRP54 by increasing its affinity for GTP. Thus, the ribosome regulates the GTPases of the SRP/SR targeting system at two stages, first after signal sequence recognition by SRP54 (
To identify components that regulate the SRß GTPase, we have used liposomes containing purified SR alone or together with translocon components, namely Sec61p complex, TRAM protein, and components of the targeting complex, namely RNC and SRP. Consistent with previous observations, we found that SR alone has a very low affinity for GTP. The apparent Kd of SR
for GTP was ~14 µM (Figure 1 B). This is in good agreement to the Kd of 10 µM, which has been previously reported (
(Kd = 20 nM) than previously reported by Miller (Kd = 1 µM;
.
Including components of the translocation machinery into the proteoliposomes with SR did not affect GTP binding to SR nor to SRß, suggesting that these components do not directly regulate the GTPases of SR. In contrast, RNCs were found to drastically reduce GTP binding to SRß. In addition, they specifically stimulate GTP hydrolysis by SRß. We suggest that the RNC induces a conformational change of the GTPase domain of SRß that leads to both an increased GTP hydrolysis and a reduced guanine nucleotide binding. As free SRß binds GTP with high affinity, interaction of SRß with the ribosome first induces hydrolysis of bound GTP, and the resulting GDP is then bound with low affinity. The low GDP affinity might increase the dissociation of the bound GDP, creating an empty state of the GTPase domain.
SRP alone or in combination with RNC showed no effect on GTP binding and hydrolysis by SRß, indicating that it functionally interacts only with SR. When SRß is associated with SR
, the presence of SRP leads to the observed burst in GTP hydrolysis via the reciprocal stimulation of GTP hydrolysis by SRP54 and SR
, which was previously shown (
The difference in regulation of SRß and SRP54/SR is in agreement with the difference in the primary GTPase domain structure of these molecules. The GTPase domains of SRP54 and SR
are related and contain an insertion box that stabilizes the nucleotide-free form of the proteins, resulting in the low affinity for GTP (
Ribosomes can bind to ER membranes independently of a nascent chain or SRP (
The interaction between ribosomes and SRß described here is unlikely to directly contribute to binding of ribosomes to the ER membrane. For this, the high affinity binding between the Sec61p complex and the ribosome is probably sufficient (
How is the GTPase cycle of SRß related to the function of the other two translocation GTPases, SRP54 and SR? The first step in targeting nascent secretory and membrane proteins to the ER membrane is the interaction of SRP with the signal sequence exposed on a ribosome (Figure 7I). The additional interaction of SRP54 with the ribosome leads to GTP binding and an activated RNCSRPGTP targeting complex (Figure 7, II;
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Because of the high affinity between ribosomes and the Sec61p complex ( to scan the ribosome for the presence of SRP and trigger the release of the signal sequence from SRP54 when GTP has been bound (Figure 7V). The dual contacts between RNC/SRP and the membrane, via an interaction between the ribosome and SRß, and SRP and SR
may ensure that only the combination of ribosomes and SRP make a functional targeting complex.
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Footnotes |
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Gerald Bacher's present address is ASTA Medica AG, Weismuellerstr. 45, 60314 Frankfurt am Main, Germany.
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Acknowledgements |
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We thank S. Hauser for generation of the antibodies against the -subunit of SR and O. Gruss for help with purifying the Sec61p complex.
This work was supported by grants from Deutsche Forschungsgemein-schaft-Schwerpunktprogramm GTPasen and the Landesforschungsschwerpunkt-Programm des Landes Baden-Württemberg.
Submitted: May 11, 1999; Revised: July 19, 1999; Accepted: July 19, 1999.
1.used in this paper: GAP, GTPase activating protein; GEFs, guanine nucleotide exchange factors; PPL, preprolactin; RNC, ribosomenascent chain complex; SR, signal recognition particle receptor; SR,
-subunit of the SR; SRß, ß-subunit of the SR; SRP, signal recognition particle
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References |
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