Article |
Address correspondence to Reid Gilmore, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation St., Worcester, MA 01605-2324. Tel.: (508) 856-5894. Fax: (508) 856-6464. email: reid.gilmore{at}umassmed.edu
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Abstract |
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Key Words: protein translocation; signal recognition particle; ribosome; endoplasmic reticulum; biosensor
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Introduction |
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Results |
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The preceding experiment suggested that RNC-bound SRP was not the active component in the GTPase assay. To address this possibility, RNCs were assembled by translation of truncated mRNAs that encode the NH2-terminal 64 residues of the G protein of vesicular stomatitis virus (pG64) and the NH2-terminal 77 residues of firefly luciferase (ffluc77). Unlike pPL86 or pG64, ffluc77 lacks a signal sequence for protein translocation across the endoplasmic reticulum; hence, SRP does not bind to ffluc77 RNCs. The GTPase activity of the SRPSR complex was stimulated by both RNC preparations (Fig. 2 C). Less than 20% of the ribosomes in a wheat germ translation reaction are assembled into RNC complexes, so the RNC preparations obtained by centrifugation contain a mixture of RNCs and nontranslating ribosomes. The GTPase assays were conducted using mock RNCs that were prepared from a translation reaction that lacked mRNA (Fig. 2 D). The GTPase cycle of SRPSR complex was stimulated by mock RNCs, suggesting that nontranslating ribosomes and free SRP are the active components in the GTPase assay.
A significant fraction of the SR in a proteoliposome faces the liposome interior and is inaccessible to the SRP and the RNCs. As observed previously (Connolly and Gilmore, 1993), the GTPase activity of the SRP and SR is low when assayed in a physiological ionic strength buffer containing detergent micelles (Fig. 3 A, open squares). The GTPase activity was stimulated roughly eightfold by the addition of pG64 RNCs (Fig. 3 A, filled squares). Deletion of SRP (Fig. 3 A, triangles) or the SR (not depicted) reduced the GTPase activity to that shown by the RNC preparation alone (circles). The GTPase activity in assays containing detergent micelles (Fig. 3 A) was higher than in assays containing the SR proteoliposomes (Fig. 2 A). Subsequent experiments used the detergent micelle assay because the GTPase activity was not influenced by experimental variations in the efficiency of proteoliposome formation or in the asymmetry of SR reconstitution.
SRP binds to nontranslating 80S ribosomes (Walter et al., 1981; Powers and Walter, 1996) in addition to polysomes synthesizing secretory proteins (Walter et al., 1981). Although earlier, nonequilibrium methods indicated that SRP binds the ribosome with a relatively low affinity (Kd 50 µM; Walter et al., 1981), a recent analysis indicates that the binding affinity is substantially higher (Kd
8 nM; Flanagan et al., 2003). Purified 80S ribosomes were added to the GTPase assays of SRP and the SR to determine whether the ribosome is the active component in the RNC preparation (Fig. 3 B, squares). Notably, half-maximal stimulation of the GTPase activity was achieved when the concentration of ribosomes exceeded the concentration of the SRP and the SR. A saturable, but much lower, stimulation of GTP hydrolysis was observed in the absence of SRP (Fig. 3 B, circles). The binding site for SRP54 on the ribosome has been mapped to ribosomal proteins L23a and L35 (Pool et al., 2002), which are located in the vicinity of the polypeptide exit site on the large ribosomal subunit (Ban et al., 2000). If the ribosome stimulates the GTPase activity of the SRPSR complex in a specific manner, one would predict that the stimulatory activity would reside on the large ribosomal subunit. Indeed, the isolated 60S subunits were almost as effective as the intact ribosome (Fig. 3 C). In contrast, 40S ribosomal subunits were comparatively ineffective even when present at a higher concentration. Neither 40S nor 60S subunits stimulated the GTPase activity of the SR in assays that were not supplemented with SRP (Fig. 3 C).
Two classes of mechanisms could explain how the ribosome could accelerate GTP hydrolysis by SRP and the SR. The addition of ribosomes could promote formation of hydrolytically active SRPSR complexes, or the ribosome could accelerate a rate-limiting step in the hydrolysis cycle without affecting the equilibrium between SRP, the SR, and the SRPSR complex. For example, a significant increase in the binding affinity of SR for GTP would increase hydrolysis because the GTPase cycle is dependent upon ribonucleotide binding to both SRP54 and SR
(Powers and Walter, 1995). Of the two GTPases (SR
and SRP54), SR
has a lower affinity for GTP, hence the Km for GTP hydrolysis provides an accurate estimate of the Kd for SR
. The Km for GTP was determined in SRPSR GTPase assays that contained or lacked 80S ribosomes. In the absence of ribosomes, the Km for GTP was 2.4 ± 1.0 µM (not depicted), which is in good agreement with a previous determination (Connolly and Gilmore, 1993). The Km for GTP was not significantly altered by the addition of ribosomes (Km = 1.4 ± 0.3 µM; not depicted), indicating that the ribosome does not stimulate the GTPase reaction by altering the nucleotide binding properties of SR
.
The interaction between the SRP and nontranslating ribosomes is reduced by increased ionic strength (Walter and Blobel, 1983; Powers and Walter, 1996). The GTPase activity of the putative ternary complex (SRSRP80S ribosome) was significantly less salt sensitive than the GTPase activity of the binary SRPSR complex (Fig. 3 D), suggesting that the ribosome stabilizes the interaction between the SRP and the SR.
GTPase activities of the SR and the SR subunits
The subunits of the SR (Fig. 4 A) were expressed in Escherichia coli to investigate the mechanism of the ribosome-stimulated GTPase activity. To facilitate purification of SRß, the lumenal and transmembrane domains of SRß were replaced with a 13-kD domain that is biotinylated in vivo to obtain bt-SRß (Fig. 4 B, lane b) or a hexahistidine sequence to obtain His-SRß (Fig. 4 B, lane e). Biotinylation domain fusion constructs were also used to express bt-SR and bt-SR
N (Fig. 4 B, lanes c and d). SR
N lacks the NH2-terminal 151 residues of SR
and corresponds to the COOH-terminal fragment of SR
that can be produced by limited digestion of microsomes with elastase (Meyer and Dobberstein, 1980; Lauffer et al., 1985). Coexpression of bt-SRß and SR
allowed the isolation of bt-SR (Fig. 4 B, lane a). Coexpression of His-SRß with NH2-terminal fragments of SR
(SR
151 or SR
319) allowed purification of His-SRßSR
151 (Fig. 4 B, lane f) and His-SRßSR
319 (Fig. 4 B, lane g). In vivo formation of the SR heterodimers (bt-SR, His-SRßSR
151, and His-SRßSR
319) provides evidence that SRß and the SRX domain of SR
are correctly folded.
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The GTPase activity of the SR purified from canine pancreas was compared with the recombinant proteins using either a hypotonic (50 mM K+) assay buffer to maximize the interaction between SRP and the SR (Fig. 4 D) or a physiological ionic strength (150 mM K+) buffer (Fig. 4 E). The E. coliexpressed proteins and the canine SR have barely detectable GTPase activities in the absence of SRP (Fig. 4, D and E). Likewise, SRP has a very low intrinsic GTPase activity (Fig. 4 E). When assayed using the low ionic strength assay buffer (50 mM K+), the bt-SR and bt-SR form complexes with SRP that hydrolyze GTP at a rate that is comparable to the SR purified from canine pancreas (Fig. 4 D). Consistent with Fig. 3 D, the GTPase activity of complexes between SRP and the SR, or SR
, was reduced in the physiological ionic strength buffer (Fig. 4 E). SRß did not hydrolyze GTP at a significant rate in the absence or presence of SRP (Fig. 4 E). The GTPase activities for SRP plus bt-SR
N showed an additive, rather than synergistic, response, indicating that active complexes were not formed between SRP and the COOH-terminal fragment of SR
(Fig. 4 E).
The E. coliexpressed SR and SR subunits were assayed for GTPase activity in the presence of 80S ribosomes (Fig. 4 F). The ability of 80S ribosomes to activate the GTPase activity of the SRPSR complex was confirmed using the E. coliexpressed bt-SR and bt-SR. Purified ribosomes did not stimulate GTP hydrolysis by bt-SRß or bt-SR
N in the presence or absence of SRP. Assays of His-SRß, His-SRßSR
151, and His-SRßSR
319 yielded results that were similar to bt-SRß (unpublished data).
The rate of SRP-SR complex formation is not accelerated by GTP
Having established that the E. coliexpressed proteins are functional by several criteria, we investigated the kinetics of SRPSR complex formation using the IAsys optical biosensor. The immobilization strategy for the SR or the SR subunits was to coat a biotin-modified biosensor cuvette with streptavidin. After removing unbound streptavidin, the sensor surface was completed by the addition of bt-SR, bt-SR, bt-SRß, or bt-SR
N. Binding of SRP to the SR or the SR subunits was initially analyzed in a hypotonic assay buffer (50 mM K+) in the absence of GTP (Fig. 5 A). SRP binds to biosensor cuvettes containing immobilized bt-SR (Fig. 5 A, a) and bt-SR
(b). SRP did not bind to bt-SR
N (Fig. 5 A, c), bt-SRß (d), or to cuvettes that contained streptavidin alone (e). The dissociation of bound SRP was monitored when applicable (Fig. 5 A, a and b). The kinetics of SRP binding to the SR was analyzed by linear regression analysis of the association curves to determine the change in refractive index caused by SRP binding (extent) and to determine the initial rate of SRP binding (kon). Hyperbolic saturation curves for binding of SRP to the SR were obtained (Fig. 5 B). The Kd value derived from the saturation curve for SRP binding to the SR in the presence of GTP is 7.6 nM, which is in reasonable agreement with the value of 15 nM that was estimated using a GTPase assay (Connolly and Gilmore, 1993). Plots of kon versus SRP concentration were linear (Fig. 5 C, filled squares). The slope and y intercept of the kon plot correspond to the rate constants for association (kass) and dissociation (kdiss), respectively, and yield a Kd value of 6.5 nM (Table I). Assay points obtained in the absence of GTP (Fig. 5 C, open squares) were adequately fit by very similar kinetic parameters (Table I). The binding kinetics of SRP to the SR was also examined in a physiological ionic strength buffer (150 mM K+) in the presence or absence of GTP (Fig. 5 C, filled and open triangles, respectively). The increase in ionic strength dramatically reduces the rate constant for complex formation, without significantly altering the rate of dissociation. The association rates for complexes formed in the absence of GTP were not significantly different from association rates obtained in the presence of GTP (Table I). The rates of dissociation were likewise not significantly influenced by the guanine nucleotide. The observation that GTP does not decrease the apparent rate constant for complex dissociation is explained by the fact that the rate of GTP hydrolysis (kcat = 3.5 x 10-2 s-1) is more rapid than kdiss for assays conducted in the absence or presence of GTP. Consequently, GTP hydrolysis is not the rate-limiting step in the dissociation reaction. The calculated Kd values for the SRPSR complex in a physiological ionic strength buffer are not significantly different from each other (Table I) and are in reasonable agreement with the Kd value (Kd + GTP = 125 nM) estimated using the GTPase assay (Connolly and Gilmore, 1993).
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Binding of ribosomes to the SR
A ternary complex between the ribosome, the SRP, and the SR might involve direct contact between the ribosome and the SR. Possible interactions between the ribosome and the SR or the SR subunits were explored using the biosensor (Fig. 6 A). Ribosomes bind to immobilized bt-SR (Fig. 6 A, a) and bt-SR (b) but not to bt-SRß (c) or to bt-SR
N (e) in the physiological ionic strength buffer (150 mM K+). The addition of GTP did not increase binding of ribosomes to SRß (Fig. 6 A, d). Large ribosomal subunits (Fig. 6 A, g), but not small subunits (h), recapitulate the binding to the SR that is observed for the intact ribosome (f). Hyperbolic binding curves for the interaction between the ribosome and immobilized SR (Fig. 6 B) yielded a Kd value of 1.9 ± 0.6 nM. The rate constants for the association (kass) and dissociation (kdiss) reactions were calculated from the kon plot (Fig. 6 B, inset) and are shown in Table I. Similar kinetic parameters were obtained for binding of the 60S subunit to bt-SR (Fig. 6 C and Table I).
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The cosedimentation assay provided a facile method to map the ribosome-binding domain in the SR (Fig. 6 D). The importance of the NH2-terminal 151 residues of SR was confirmed using the cosedimentation assay, as bt-SR
binds to ribosomes (Fig. 6 D, g and h) but bt-SR
N does not (e and f). Neither tagged derivative of SRß cosedimented with 80S ribosomes (Fig. 6 D, il). The NH2-terminal 151 residues of SR
are not sufficient for ribosome-binding activity even when coexpressed with His-SRß (Fig. 6 D, o and p). In contrast, His-SRßSR
319 was recovered in the pellet fraction when ribosomes were included (Fig. 6 D, m and n).
The conventional assay used to characterize the ribosome-binding activity of purified ER membrane proteins monitors binding of radiolabeled ribosomes to proteoliposomes (Kalies et al., 1994; Raden et al., 2000, and references therein). The purified canine SR was reconstituted into proteoliposomes and incubated with various quantities of 125I-labeled ribosomes in a physiological ionic strength buffer (150 mM K+). Proteoliposome-bound and unbound ribosomes were separated by gel filtration chromatography to obtain a saturation curve (Fig. 7 A). The apparent Kd value obtained in this experiment (0.87 ± 0.02 nM) is in good agreement with the Kd value obtained using the biosensor. More importantly, the stoichiometry of binding was found to be roughly 1:1 based upon the experimentally determined Bmax value and the concentration of SR in the proteoliposomes. Binding of 80S ribosomes to Sec61 proteoliposomes was analyzed as a control (Fig. 7 B), and the Kd value we obtained (5.4 ± 0.9 nM) was in good agreement with the previous literature (Kalies et al., 1994).
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Discussion |
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Previous studies using canine microsomes or SR proteoliposomes have indicated that GTP is not required for targeting of SRPRNCs to the SR (Rapiejko and Gilmore, 1994; Song et al., 2000), and that the targeting step precedes cooperative, stable binding of GTP to SRP54 and SR (Rapiejko and Gilmore, 1997). Nonetheless these previous studies do not eliminate the formal possibility that the affinity of the SRPRNC for the SR could be enhanced by rapidly reversible low-affinity binding of GTP to the empty site forms of SRP54 and SR
. As shown here, biosensor experiments designed to monitor the binding affinity between SRP and the SR demonstrated that the addition of GTP did not significantly increase the rate constant for formation of the SRPSR complex in either isotonic or hypotonic buffers. As reported previously (Connolly et al., 1991), the nonhydrolyzable GTP analogue Gpp(NH)p stabilizes the SRPSR complex by reducing the dissociation rate.
Could signal sequencespecific binding of SRP to an RNC cause a conformational change in SRP that enhances the affinity between SRP and the SR? This hypothesis was based upon the report that RNCs assembled in an in vitro translation system activate the GTPase activity of the SRPSR complex (Bacher et al., 1996, 1999). Our analysis of this experimental system disclosed the remarkable finding that nontranslating ribosomes activate the GTPase activity of the SRPSR complex. Furthermore, bona fide SRPRNC complexes assembled using a secretory mRNA did not hydrolyze GTP when targeted to the SR proteoliposomes, consistent with our previous observation that both GTP and Gpp(NH)p stabilize the SRSRPRNC complex (Song et al., 2000).
The third hypothesis we considered was that the ribosome forms a platform for assembly of the SRPSR complex. Purified 60S ribosomal subunits, but not 40S ribosomal subunits, stimulate the GTPase activity of the SRPSR complex, consistent with the evidence that SRP54 binds to the L23a and L35 proteins in the large ribosomal subunit (Pool et al., 2002). Here, we obtained evidence that the SR has a high binding affinity for purified ribosomes or 60S ribosomal subunits. Notably, the rate constant for association of a ribosomeSR complex is 300-fold faster than the rate constant for formation of the SRPSR complex. Consequently, the kinetics of targeting of the SRPRNC complex to the SR should be dominated by the SRribosome interaction. The SRribosome interaction is also characterized by a relatively fast dissociation rate (kdiss 1.2 x 10-2 s-1). The rate of dissociation of SRP from the SR is less rapid, and this rate should decrease for the SRPRNC complex. Binding of GTP to SRP54 and SR
substantially increases the stability of the SRPRNCSR complex because GTP hydrolysis by SRP54 and SR
is delayed until a vacant Sec61 complex is identified as an acceptor for the RNC complex (Song et al., 2000). We propose that the SR, by dual recognition of the ribosome and the SRP, will reject ribosomes that lack bound SRP.
The affinity between the SR and the ribosome appears to be conserved between eukaryotic and prokaryotic organisms. Depletion of either the translocon subunit SecE or the bacterial SRP (Ffh) leads to the in vivo accumulation of membrane-bound ribosomeFtsY complexes (Herskovits et al., 2002). The NH2-terminal acidic (A) domain of FtsY, which is involved in membrane binding (de Leeuw et al., 1997), is not homologous to the NH2-terminal 319 residues of SR. Further work will be required to define the structural basis for the evolutionarily conserved interaction between the ribosome and the SR.
Roles for the SR subunits
The SRß subunit of the SR was dispensable for the GTPase activity of the SRPSR complex. Complexes formed between FtsY, the prokaryotic equivalent of SR, and Ffh4.5S RNA, the prokaryotic equivalent of SRP54 and the 7S RNA, hydrolyze GTP in a cooperative manner that has been investigated as a paradigm for the SRPSR complex (Powers and Walter, 1995; Jagath et al., 2000; Peluso et al., 2000), hence it was not surprising that SRß was dispensable for the GTPase cycles of SRP54 and SR
.
The observation that SRß does not hydrolyze GTP when assayed alone was not unexpected, as most GTPases have very low hydrolysis rates in the absence of GEFs and GTPase-activating proteins (GAPs) (Bourne et al., 1991). SRß does not hydrolyze GTP in the presence of 80S ribosomes, indicating that the ribosome cannot fulfill both the GEF and GAP functions for SRß. Although photolabeling experiments had suggested that the ribosome acts as a GEF to stabilize a nucleotide-free form of SRß (Bacher et al., 1999), a more recent report does not support this conclusion (Legate and Andrews, 2003). Our GTPase assays do not address which step, or steps, in the SRß GTPase cycle occurs in the presence of the ribosome.
The SRX domain of SR (residues 1178) is necessary and sufficient for GTP-dependent heterodimerization with SRß (Young et al., 1995; Ogg et al., 1998; Legate et al., 2000; Schwartz and Blobel, 2003). The GTP-bound, but not GDP-bound, form of SRß forms stable heterodimers with SRX (Schwartz and Blobel, 2003). In the absence of a currently unidentified SRß GAP, the SRß GTPase is thought to be catalytically inert when bound to SRX (Schwartz and Blobel, 2003). An alternative model for the SRß GTPase cycle proposes that GTP binding to SRß regulates the release of the signal sequence from SRP54 (Fulga et al., 2001).
SRß can be cross-linked to a 21-kD protein in the large ribosomal subunit (Fulga et al., 2001). Here, we observe an SRß-independent, high-affinity interaction between SR and the 60S subunit, suggesting that SR
positions SRß adjacent to the 21-kD protein. As bt-SR
and bt-SR have similar affinities for the ribosome, we conclude that SRß does not occlude the ribosome-binding site on SR
nor does it enhance the affinity of the SR for the ribosome. Previous studies that analyzed the ribosomeSRß interaction have used either the recombinant SR heterodimer (Fulga et al., 2001) or trypsin-digested SR heterodimers that retain the NH2-terminal fragment of SR
(Bacher et al., 1999). The discrepancy between our results and these previous studies concerning the ribosome-binding and GTPase activities of SRß might be explained by these structural differences in the reagents.
The NH2-terminal domain of SR that is sufficient for ribosome-binding activity is polar (64% charged or polar residues) and basic (pI = 9.16). GTPase assays and biosensor experiments showed that SRP does not bind to bt-SR
N, despite the evidence that bt-SR
N is properly folded. It is unlikely that the NH2-terminal 151 residues of SR
are sufficient for the interaction of the SR with SRP, as the GTPase cycle of the SRPSR complex almost certainly requires direct contact between the N and G domains of SRP54 and SR
. A model for the FfhFtsY complex (Montoya et al., 2000) predicts important interactions between the G domains of the two GTPases.
Regulation of the SRribosome interaction
Within the cell, a futile GTPase cycle catalyzed by the SRPSR complex is not favored due to the low affinity between the SR and free SRP. However, the discovery that the ribosome can promote assembly of the SRPSR complex in isotonic buffers raises new questions about the in vivo regulation of the SRPSR GTPase cycle. A futile cycle involving SRP, the SR, and a ribosome would be restricted to the RER surface and would depend upon the presence of SRP and SR that are not engaged in bona fide targeting reactions. The cellular concentration of SRP and the SR may be regulated to ensure that the GTPases are substoichiometric relative to membrane-bound ribosomes.
Nontranslating ribosomes do not compete with SRPRNCs for targeting to the Sec61 complex (Raden and Gilmore, 1998). This observation strongly suggests that there must be a mechanism to prevent the SR from being saturated by 60S ribosomal subunits, or ribosomes that are not engaged in the synthesis of secretory proteins. Although the relatively rapid dissociation rate for the ribosomeSR complex may contribute to such a mechanism, we speculate that there are additional factors that destabilize the SRribosome complex by selectively accelerating the dissociation rate. Ribosomes bearing nascent polypeptides that are synthesized on cytoplasmic polysomes recruit ribosome-associated chaperones, including the nascent chainassociated complex and members of the Hsc70 family (Wang et al., 1995; Bukau et al., 2000). A role for the nascent chainassociated complex in preventing signal sequenceindependent binding of RNCs to the translocation channel has been proposed (Lauring et al., 1995; Moller et al., 1998), but the mechanism remains a matter of controversy (Neuhof et al., 1998; Raden and Gilmore, 1998). Future experiments will address the possibility that cytosolic chaperones regulate the binding affinity between the SR and the ribosome.
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Materials and methods |
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Isolation of ribosomes, ribosomal subunits, and RNC complexes and radioiodination of ribosomes
Ribosomes were isolated from canine RM by extraction with high salt as previously described (Collins and Gilmore, 1991). Residual SRP was separated from 80S ribosomes by two sequential centrifugations through a high saltsucrose cushion (Collins and Gilmore, 1991) followed by centrifugation through a physiological saltsucrose cushion and resuspension of the ribosomes in buffer A (50 mM triethanolamine-acetate [TEA], pH 7.5, 150 mM KOAc, 5 mM Mg[OA]2, 1 mM DTT). Canine 80S ribosomes were dissociated into 40S and 60S subunits by treatment with 1 mM puromycin in buffer A, after which the sample was applied to a 14-ml 1030% sucrose gradient in 50 mM TEA, 500 mM KOAc, 5 mM Mg(OAc)2, 1 mM DTT. The ribosomal subunits were resolved by centrifugation for 4.5 h at 200,000 gav using a Beckman Coulter SW40 rotor.
Sucrose gradientpurified 80S ribosomes (Raden et al., 2000) were resuspended in DTT-free buffer A and labeled with 125I Bolton-Hunter reagent (Amersham Biosciences) as previously described (Raden et al., 2000). Proteoliposomes were prepared as previously described (Song et al., 2000) using a modification of the method of Görlich and Rapoport (1993). Binding of 125I-labeled ribosomes to proteoliposomes was assayed as previously described (Raden et al., 2000). In brief, 0.030.65 pmol of 125I-labeled ribosomes was incubated with aliquots of the proteoliposomes in buffer A. The 25-µl sample was applied to a 1.2-ml Sepharose CL-2B column equilibrated in buffer A to resolve proteoliposome-bound ribosomes (0.30.6 ml of eluate) from unbound ribosomes (0.61.5 ml of eluate).
Truncated mRNAs encoding the NH2-terminal 86 residues of pPL (pPL86), 64 residues of vesicular stomatitis virus glycoprotein (pG64), 77 residues of firefly luciferase (ffluc77), or 156 residues of bovine opsin (op156) were prepared as previously described (Rapiejko and Gilmore, 1994).
SRPRNCop156 complexes were assembled in a reticulocyte lysate reaction as previously described (Rapiejko and Gilmore, 1997) and adjusted to 375 µM cycloheximide to block further translation. Membrane integration and N-linked glycosylation of op156 were assayed as previously described (Rapiejko and Gilmore, 1997).
RNC complexes bearing pPL86, pG64, or ffluc77 were assembled by translating truncated mRNAs for 15 min in a wheat germ reaction that lacked radiolabeled amino acids and SRP, unless noted otherwise. After blocking further translation by the addition of 2 mM cycloheximide, the translation products were adjusted to 500 mM KOAc before centrifugation for 1 h at 400,000 gav at 4°C through a high saltsucrose cushion (1 M sucrose, 25 mM Hepes-KOH, pH 7.8, 500 mM KOAc, 5 mM Mg(OAc)2, 1 mM cycloheximide, 1 mM DTT). The RNCs were resuspended in half of the volume of the translation reaction in buffer B (25 mM Hepes-KOH, pH 7.8, 5 mM Mg[OAc]2, 1 mM cycloheximide, 1 mM DTT) adjusted to 500 mM KOAc and reisolated by centrifugation as described above. Finally, the RNCs were resuspended in buffer B adjusted to 150 mM KOAc at a concentration of 1 µM ribosomes.
GTPase assays
GTPase assays were conducted at 25°C in a total volume of 5 µl and contained 2550 nM SR (canine SR, recombinant SR, or SR subunits), 50 nM SRP, 140 nM RNCs or mock RNCs, and 0.5 µM [-32P]GTP (410 Ci/mmol) in buffer C (50 mM TEA-OAc, 150 mM KOAc, 5 mM Mg[OAc]2, 2 mM DTT, 2 mM cycloheximide) unless noted otherwise. The detergent micelle GTPase assays contained 0.1% Nikkol. Aliquots of the GTPase assays were removed at frequent time intervals and spotted onto PEI-cellulose thin layer plates to resolve GDP from GTP (Connolly and Gilmore, 1993).
Expression and purification of SR and SR subunits
DNA encoding a canine SRß derivative lacking the NH2-terminal 54 residues (SRßN) was obtained by PCR amplification of the SRß plasmid pMAC455 (Young et al., 1995) using appropriate primers and standard PCR conditions. The SRß
N coding sequence was inserted into the PinPoint vector (Promega) to obtain pbt-SRß
N. The dicistronic plasmid pbt-SRß-SR
encoding bt-SRß and SR
was constructed by inserting the SR
coding sequence derived from plasmid pG4
(Rapiejko and Gilmore, 1992) into pbt-SRß
N. The plasmids pbt-SR
and pbt-SR
N encode fusion proteins between the biotinylation domain and canine SR
or canine SR
lacking the NH2-terminal 151 residues, respectively. The SRß
N sequence was subcloned into pET14b (Novagen) to obtain pHis-SRß. Heterodimers consisting of His-SRß and NH2-terminal fragments of SR
(SR
151 or SR
319) were expressed from dicistronic plasmids. All constructs were verified by DNA sequencing. The biotinylated proteins were purified from the E. coli (J109) lysates by affinity chromatography (Soft-Avidin resin; Promega) and anion and cation exchange chromatography. The His-tagged proteins were purified from E. coli (Rosetta; Novagen) lysates by Ni-NTA (QIAGEN) affinity chromatography and cation exchange chromatography.
IAsys affinity sensor experiments
Binding of SRP or ribosomes to the SR or SR subunits was assayed using an IAsys affinity sensor (Affinity Sensors). The binding surface was constructed by incubating saturating amounts of streptavidin (Promega) with a biotin-coated cuvette for 5 min. After a brief wash with buffer D (50 mM TEA, 150 mM KOAc, 2.5 mM Mg[OAc]2, 0.1% Nikkol) the bt-SR or a bt-SR subunit was added and incubated until equilibrium binding was observed (5 min). Preparation of the sensor surface was followed by a brief wash with buffer D. Binding time courses were performed at 25°C using a variety of ligates (SRP, ribosomes, or ribosomal subunits) in buffers D or E (buffer D with KOAc reduced to 50 mM) in either the absence or presence of 25 µM GTP. The ligate was preincubated for 2 min at 25°C (with GTP when appropriate) before the addition to a cuvette containing the immobilized bt-protein. Analysis of binding experiments showed that 6.5 min was sufficient to calculate equilibrium binding values. After binding, the cuvette was rapidly washed three times with buffer D or E (with or without GTP), and dissociation of ligate was monitored for 3 min. The binding surface was regenerated by dissociating residual ligate with buffer F (50 mM TEA, 500 mM KOAc, 5 mM Mg[OAc]2, 0.1% Nikkol). The cuvette was then washed and reequilibriated in buffer D or E containing GTP as indicated. The high salt wash procedure removes the ligate without damaging or detaching the bt-protein. Dissociation of Gpp(NH)pstabilized bt-SRSRP complexes was incomplete.
Binding of ligate to the sensor surface is measured as a response (arc seconds of change in the refractive index), which corresponds to the accumulation of mass within the optical window at the binding surface. The extent (in arc seconds) refers to the calculated maximum response (Rmax) at equilibrium for a given concentration of ligate. The rates of ligate binding (kon) and the extent (Rmax) were calculated from association curves using FASTfit software supplied with the instrument.
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Acknowledgments |
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This work was supported by National Institutes of Health grant PHS GM35687.
Submitted: 24 March 2003
Accepted: 30 June 2003
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