GTPase cycle and ER membrane regulate cargo handover during cotranslational protein targeting

Abstract

Signal recognition particle (SRP), a universally conserved protein-targeting machine, cotranslationally recognizes signal sequences of nascent endomembrane proteins and engages the SRP receptor (SR) at the endoplasmic reticulum (ER) membrane to unload the translating ribosome. The mechanism of membrane-associated steps in this pathway remains unclear. Here, we address this question using pre–steady-state and single-molecule fluorescence measurements. SR induces release of the signal sequence from SRP and detachment of the SRP guanosine triphosphatase (GTPase) domain from the ribosome. Despite the dissolution of these contacts, SR acts as a tether to stabilize SRP association with the ribosome. Guanosine 5′-triphosphate (GTP) hydrolysis in the SRP-SR complex abolishes this stabilizing effect to facilitate ribosome-nascent chain complex (RNC) release from SRP. The ER membrane selectively promotes a prehandover conformation of the RNC-SRP-SR complex, in which delayed GTP hydrolysis optimizes the transition from targeting to translocation. Our findings provide a high-resolution model for key steps during cargo handover at the ER and highlight the critical role of fine-tuned GTPase activity in the SRP pathway.

INTRODUCTION

Membrane and secretory proteins destined to the endomembrane system comprise over 30% of the newly synthesized proteome in eukaryotic cells and are initially delivered to the endoplasmic reticulum (ER). To minimize misfolding and aggregation in the cytosol, their ER targeting is mediated cotranslationally by signal recognition particle (SRP), which delivers translating ribosomes to the ER via interaction with the SRP receptor (SR). At the ER membrane, SRP hands over translating ribosomes to protein translocation machineries, including the Sec61p complex and ER membrane complex (EMC), via which nascent proteins are inserted into or translocated across the membrane (1–3). Disruption of SRP function leads to mislocalization of proteins, mitochondrial stress, and disruption of proteostasis (4–6). Mutations in SRP are linked to devastating diseases including severe congenital neutropenia and Shwachman-Diamond–like syndromes, manifesting in reduced granulocyte maturation, bone marrow failure, and developmental disorders (4–6).

Eukaryotic SRP is a ribonucleoprotein complex composed of the 7SL RNA tightly bound to six protein subunits (SRP9, SRP14, SRP19, SRP54, SRP68, and SRP72) (7). The methionine-rich M-domain of SRP54 is positioned at the ribosomal exit tunnel, where it recognizes ER-targeting signals on the nascent protein, typically a transmembrane domain (TMD) or a hydrophobic signal sequence near the N terminus, as they emerge from the ribosome (8–10). Signal sequence recognition drives SRP into a “proximal” conformation, in which a special guanosine triphosphatase (GTPase), NG-domain in SRP54 interacts with ribosomal proteins uL23 and uL29 near the tunnel exit (8). Additional ribosome contacts are made by SRP68/72 at the distal site of SRP and by SRP9/14 at the 40S to 60S interface (8). The multidentate interactions result in high-affinity recognition of the ribosome-nascent chain complex (RNC), with equilibrium dissociation constants (K_d) in the subnanomolar range (11, 12).

Recognition of ribosomes bearing an ER-targeting signal activates SRP for efficient binding with SR, a heterodimer of SRα and SRβ subunits in eukaryotic cells (13–15). SRβ anchors SR to the ER membrane via its TMD, while its cytosolic domain mediates stable dimerization with the N-terminal X-domain in SRα (16, 17). A 170–amino acid–long linker connects the membrane proximal X-domain of SRα to its C-terminal NG-domain, which is highly homologous to the NG-domain in SRP54. SRP-SR association initiates with the guanosine 5′-triphosphate (GTP)–dependent dimerization of their NG-domains (14, 18) and is followed by sequential conformational rearrangements, in which the NG-dimer detaches from the ribosome exit site and docks instead at SRP68/72 together with SRαX and SRβ (Fig. 1A). This generates a “prehandover” conformation of the targeting complex in which the RNC is brought closer to the ER membrane, and the uL23/29 site on the ribosome is vacated for subsequent docking at the Sec61p translocation machinery (9, 13, 19). Mutations that disrupt the formation of the prehandover conformation impaired targeting efficiency (5, 13), supporting the importance of this conformation. Last, reciprocal GTPase activation occurs in the SRP-SR NG-dimer, and GTP hydrolysis drives the dissociation of SRP from SR (14, 15, 18, 20).

Fig. 1. SR induces signal sequence release from the SRP54 M-domain.

(A) Structure of the prehandover RNC-SRP-SR complex [PDB: 6FRK (19)]. The ribosome is in gray. SRP54 (cyan), SRα (light green), SRβ (dark green), SRP68 (teal), and the rest of SRP (tan) are shown in surface representation. (B) Fluorescence emission spectra for the indicated samples. SRP*, SRP^M-Cy3B; RNC*, RNC^ATTO647N. Where indicated, 2 nM SRP*, 200 nM SR, 100 μM GMPPNP, and 10 nM RNC* or unlabeled RNC were used. To compete off the FRET signal, 100 nM unlabeled RNC (10×) was added to a preformed SRP*-RNC* complex (green). (C) Scheme of the smTIRFM assay to monitor the distance between signal sequence and SRP54 M-domain. Green and red stars depict the donor and acceptor dyes on SRP^M-Cy3B and RNC^ATTO647N, respectively, which gains high FRET when the signal sequence docks at the SRP54 M-domain. SR-induced conformational rearrangements of SRP are shown, with the right panel depicting the prehandover conformation. (D) smFRET histograms of SRP bound to RNCs at different nascent chain lengths (L), with (purple bars) and without (black line) SRαβΔTM present. N is the number of frames used to construct each histogram. The data in the presence of SR were fit to the sum (solid outline) of three Gaussian distributions with low, medium, and high mean FRET efficiencies (dashed lines), with the vertical lines indicating the mean FRET value of each state. The data without SR are from (11) and shown for comparison. Number of trajectories used to analyze FRET efficiency: 48 [91 amino acids (aa)], 54 (131 amino acids), 48 (151 amino acids), and 47 (171 amino acids). (E) Summary of the relative population of SRP in the low, medium and high FRET states for SRP bound to RNC with different nascent chain lengths with and without SR.

Despite extensive progress on our understanding of the mammalian SRP pathway, many questions remain about how the targeting complex transfers the RNC to the Sec61p translocase, which also binds ribosome at uL23/29 and with the signal sequence/TMD on the nascent protein. First, although the SRP NG-domain detaches from the ribosome exit site upon its dimerization with SR (13), how the signal sequence is transferred from the M-domain of SRP54 to translocation machineries is unclear. Second, nearly 100 Å separates the exit tunnel of the ribosome and the ER membrane even in the prehandover conformation (Fig. 1A) (19). Although this distance can be dependent on the orientation of the SRP-SR complex at the membrane, this observation raises questions about how Sec61p and other translocases initiate contact with SRP. While an RNC can dock on translocation machineries after it completely dissociates from SRP and SR, such a mechanism is unsatisfactory, risking loss of cargo and abortive targeting. Last, while a recent study identified the TMEM208 complex as a factor that accelerates signal sequence release from SRP, TMEM208 appears to selectively affect the biogenesis of multipass membrane proteins, whereas it has little or no effect on most single-pass membrane proteins and secretory proteins, including the canonical SRP substrate preprolactin (pPL) (21).

Furthermore, the role of GTP hydrolysis in the SRP pathway has remained elusive. It is well established that GTP hydrolysis, which drives disassembly of the SRP-SR NG-dimer, allows SRP to be recycled for multiple rounds of targeting (14, 15, 18, 20). On the other hand, GTP hydrolysis was not considered important for the targeting reaction per se, based on the cosedimentation of RNCs with ER microsomes in the presence of nonhydrolyzable GTP analogs such as guanosine 5′-[β,γ-imido]triphosphate (GMPPNP) (18). However, the cosedimentation studies measured the delivery of RNC-SRP complex to the ER but did not directly monitor RNC handover. The poor temporal resolution of cosedimentation assays (10 to 20 min) also precludes the detection of kinetic differences between GTP and GMPPNP for cargo handover, which must occur on a faster timescale during cotranslational protein targeting and translocation. Notably, facile handover of the RNC is important, as the nascent chain can lose translocation competence if it becomes excessively long before engagement with the translocase or insertase (11, 12, 22–25).

In this study, we dissected the mechanism of cargo release from SRP using pre–steady-state measurements of RNC-SRP interaction and single-molecule analyses of the conformational dynamics of SRP in the targeting complex. Our results revealed dual roles of SR in cargo handover, uncovered a role of the ER membrane in promoting the prehandover complex in a signal sequence–specific manner, and highlighted critical roles of precisely timed GTP hydrolysis in the SRP pathway. These findings established a high-resolution mechanistic model for RNC release that completes the cargo handover in SRP targeting pathway.

RESULTS

SR induces signal sequence release from the SRP54 M-domain

We recently developed a sensitive and robust assay to specifically detect the interaction of the SRP54 M-domain with signal sequence on the RNC based on Förster resonance energy transfer (FRET) between a donor dye, Cy3B, labeled at an engineered Cys³⁶³ in the SRP54 M-domain (SRP^M-Cy3B) and an acceptor dye, ATTO647N, labeled at residue 12 preceding the signal sequence of the model SRP substrate pPL on the RNC (RNC^ATTO647N) (fig. S1). The estimated distance between the FRET pair is 35 Å in the structure of the RNC_pPL-SRP complex [Protein Data Bank (PDB): 7QWQ; fig. S1] (26), compared to their Förster radius of ∼65 Å, predicting a high FRET efficiency when the signal sequence docks at the hydrophobic groove in the M-domain (11). This dye pair exhibited ∼50% FRET efficiency with the 91–amino acid–long RNC_pPL in steady-state fluorescence measurements (Fig. 1B) (11). To further probe the dynamics of signal sequence interaction with SRP, we carried out single-molecule total internal reflection fluorescence microscopy (smTIRFM; Fig. 1C) experiments in which RNC^ATTO647N was immobilized on quartz coverslips and SRP^M-Cy3B was detected only when it binds the surface-immobilized RNC (Fig. 1C). Three FRET states with high, medium, and low mean FRET efficiency were detected in hidden Markov modeling (HMM) of the smFRET traces in the absence of SR (11). The FRET histogram for the RNC-SRP complex is dominated by the high FRET population [82%; Fig. 1D, black line, L = 91, and (11)]. Mutation of two leucines in the pPL signal sequence to arginine (LLLRRVVSNLLL; termed 2R) reduced the high FRET population to 15%, with a corresponding increase in the low FRET population and a 61-fold reduced RNC-SRP binding affinity (figs. S1, B to D, and S2C). These results corroborated the assignment of the high FRET state to the docking of signal sequence at the SRP M-domain during cargo recognition by SRP.

Using this smTIRFM setup, we asked how SR affects the interaction of signal sequence with the SRP M-domain. The RNC-SRP-SR complex was assembled in GMPPNP using a soluble SR complex in which the TMD in SRβ is removed (SRαβΔTM, referred to as SR henceforth, whereas wildtype SR on the RM is referred to as endogenous SR). SRαβΔTM is highly active in mediating SRP-SR association, SR-induced conformational rearrangements in SRP, and cotranslational protein targeting (27, 28). We first verified that FRET between the dye pair could still be detected after assembly of the RNC_pPL-SRP-SR complex in steady-state measurements (Fig. 1B). In smTIRFM measurements, SR induced a substantial shift in the FRET histogram, reducing the high FRET population from 82% in the SRP-RNC complex to ∼20% in the RNC-SRP-SR complex, with corresponding rises in the population of medium- and low-FRET states (Fig. 1, D and E, L = 91). In addition, SR reduced the dwell time of SRP in the high-FRET state (fig. S3A), indicating reduced kinetic stability of this state. Together, these results show that SRP-SR association destabilizes the interaction of the signal sequence with SRP, inducing its undocking from the M-domain.

We recently reported that signal sequence docking at the SRP M-domain becomes less frequent as the nascent chain elongates (Fig. 1D, black lines) (11). To test whether the length-dependent effect also occurs in the RNC-SRP-SR complex, we varied the length of the nascent chain (L) from 91 to 171 amino acids. In the presence of SR, increasing nascent chain length beyond 131 amino acids further reduced the population and lifetime of the high FRET state ∼2-fold (Fig. 1, D and E, and figs. S2A and S3A), suggesting that a longer nascent chain modestly destabilizes signal sequence docking at the SRP M-domain in the RNC-SRP-SR complex. In addition, elongation of the nascent chain progressively increased the population of the low FRET relative to medium FRET state (Fig. 1, D and E, and fig. S2A), suggesting increased spatial separation between the signal sequence and the SRP M-domain. Nevertheless, SR is dominant in driving the release of signal sequence from the SRP M-domain at short (91 to 131 amino acids) nascent chain lengths before it becomes too long and loses targeting competence (11, 12, 22–25). Both the population and the dwell time of the medium FRET population decreased as the nascent chain elongates, whereas the mean FRET efficiency of this population remained constant (Fig. 1, D and E, and fig. S3B). These observations suggest that the medium FRET state arose from the interaction of signal sequence with an alternative site located 50 to 60 Å away, potentially on the ribosome near the polypeptide tunnel exit (29), upon its undocking from the M-domain.

SRP-SR association is accompanied by sequential conformational rearrangements in which the NG-dimer detaches from the ribosome and docks at the distal site of SRP (Fig. 1C and fig. S4A) (13). To test whether these rearrangements are required for disrupting the signal sequence interaction of SRP, we tested two established mutants. SRP54 Gly²²⁶→Glu(G226E) blocks the detachment of the NG-dimer from uL23/29 at the ribosome exit site; SRαβΔTM harbors a deletion at SRα residues 572 to 581 [SR(Δ572 to 581); table S1], which impairs the interaction of the NG-dimer with SRP68/72 in the prehandover conformation (13) (fig. S4A). SR-induced release of signal sequence from the SRP M-domain was observed with both mutants (figs. S2B and S4), indicating that SRP-SR NG-dimer assembly is sufficient to destabilize the signal sequence interaction with SRP.

These results show that SRP-SR association and nascent chain elongation work in concert to destabilize signal sequence docking at the SRP M-domain. Together with the SR-induced detachment of the SRP54 NG-domain from its uL23 docking site on the ribosome (21), both key stabilizing contacts of SRP with the RNC during their initial recognition are dissolved upon formation of the RNC-SRP-SR complex.

SR tethers SRP to the RNC via bidentate interactions

The results above suggest that SR, by displacing the signal sequence from the SRP M-domain and the SRP54 NG-domain from the ribosome, could facilitate the release of nascent protein from SRP. To test this possibility, we measured and compared the dissociation rate constant of SRP^M-Cy3B from RNC^ATTO647N in RNC-SRP and RNC-SRP-SR complexes using unlabeled SRP or SRP-SR as chase (Fig. 2A). The nonhydrolyzable GTP analog GMPPNP was used to mimic the targeting complex before GTP hydrolysis. In the absence of SR, SRP-RNC dissociation displayed biphasic kinetics, with a slow (k_off = 0.0046 s⁻¹) and a fast (k_off = 0.017 s⁻¹) phase at 1:2 ratio, yielding an overall observed dissociation rate constant of ∼0.01 s⁻¹ (Fig. 2, B and C, blue; fig. S5; and table S2). Unexpectedly and in contrast to the simplest expectation from the data in Fig. 1, complex formation with SR hindered SRP dissociation from the RNC. The dissociation kinetics of RNC-SRP-SR is single-phasic with a k_off of 0.0013 s⁻¹ (Fig. 2, B and C, orange; fig. S5; and table S2), ∼10-fold slower than that without SR. Thus, while the smTIRFM assay showed increased excursion of the signal sequence from the SRP54 M-domain within an assembled RNC-SRP-SR complex, the pulse-chase assay, which reports on the complete dissociation of SRP from the RNC, indicated that SR stabilizes overall SRP-RNC interaction.

Fig. 2. SR stabilizes SRP-RNC association.

(A) Scheme of the pulse-chase assay to measure the dissociation of SRP^M-Cy3B from RNC^ATTO647N using excess unlabeled SRP as chase. (B) Time traces of SRP dissociation from RNC-SRP or RNC-SRP-SR complexes assembled with the indicated SRαβΔTM (SR) variants in GMPPNP. Donor fluorescence intensity was normalized such that F equals 0 and 1 at the beginning and end of the reaction, respectively. The lines show the mean of three technical replicates, and gray shows the upper and lower limits across technical replicates (fig. S5). The data without SR and with SR(KR/GS) were fit to double exponential functions (Eq. 4), and the data with wild-type (WT) SR and SR(Δ572 to 581) were fit to single exponential function (Eq. 5). The fitted values are summarized in table S2. (C) Summary of the half-times of SRP dissociation from RNC without and with the indicated SR variants. (D) Top: Domain organization of SRα showing the location of the KR/GS mutations. Bottom: The KR/GS mutation impaired the binding of SR with ribosome, measured by cosedimentation assay. SRα, SRβ, and ribosomal protein bands are indicated. (E) The KR/GS mutations in the SR linker impaired cotranslational protein targeting to ER microsomes. Top: Representative SDS-PAGE autoradiograph of pPL targeting and translocation in the presence of 20 nM SRP, TKRM (0.5 eq/μl), and indicated concentrations of wild-type SR or SR(KR/GS). pPL and translocated prolactin (PL) bands are indicated. Bottom: Quantification of translocation efficiency based on cleavage of the signal sequence (Eq. 1). Data are shown as means ± SD, with n = 3 technical replicates. The lines are fits of the data to Eq. 3. (F) Model for the dual roles of SR in destabilizing signal sequence interaction with SRP while stabilizing SRP-RNC association via the K/R-rich linker.

We next sought to understand the mechanism underlying this stabilizing effect. We first hypothesized that this stabilization arises from alternative contacts of SRP in the prehandover complex, in which the SRP-SR NG-dimer forms a network of interactions with SRP68, the SRα X-domain, and SRβ (Fig. 1A) (13, 19). To test this hypothesis, we examined SR(Δ572 to 581) described above, which impairs docking of the NG-dimer at the distal site (fig. S4A). SR(Δ572 to 581) delayed RNC dissociation from SRP as strongly as wild-type SR (Fig. 2, B and C, green; fig. S5; and table S2), arguing against a contribution of the distal site interactions to SR-induced stabilization of SRP.

It was reported that the linker connecting the NG- and X-domains of SRα contains an evolutionarily conserved, positively charged K/R-rich region that can mediate interaction with the ribosome (27, 30). We therefore considered an alternative hypothesis: SR acts as a molecular tether that simultaneously interacts with the ribosome and the SRP54 NG-domain. To test this model, we replaced the K/R-rich regions in the SR linker with G/S linkers of the same length [SR(KR/GS) in table S1; Fig. 2D, top). Cosedimentation analysis confirmed that the KR/GS mutation disrupted the binding of SR with the ribosome (Fig. 2D). Pulse-chase measurements showed that this mutation reduced the stabilizing effect of SR on RNC-SRP association, repopulating the fast-dissociating phase to ∼53% and accelerating the overall dissociation of SRP-SR from RNC ∼3-fold (Fig. 2, B and C, red; fig. S5; and table S2). These results indicate that the ribosome interaction of the SR linker is responsible, at least in part, for the hyperstabilization of SRP on the RNC following its binding with SR.

We further assessed the protein-targeting activity of SR(KR/GS) using a reconstituted assay, which measures the ability of purified SRP and SRαβΔTM to target pPL, during continuous protein synthesis, to salt-washed, trypsinized ER microsome (TKRM) that retains the Sec61p translocon but is devoid of endogenous SR (27, 31). Translocation of pPL across the ER membrane was measured by cleavage of its signal sequence to yield prolactin (PL; Fig. 2E). Compared to wild-type SR, SR(KR/GS) showed a strong defect in pPL translocation at low concentrations, but the defect was rescued at higher SR concentrations (Fig. 2E). Analysis of the SR concentration dependence showed an 100-fold increase in K_m (101 nM versus 1.0 nM with mutant versus wild-type SRαβΔTM, respectively) but not in maximal translocation efficiency at saturating SR concentrations (table S3). This implies that the translocation defect stems from impaired intermolecular interactions of SR(KR/GS), consistent with its weakened ribosome interaction.

Together with the smFRET data in the previous section, these findings support a dual role of SR in modulating the RNC-SRP interaction (Fig. 2F). On the one hand, SR dissolves the contacts of SRP with both the uL23/uL29 ribosomal proteins and the signal sequence, exposing contact sites required for subsequent docking of the RNC on Sec61p. On the other hand, SR tethers SRP to the RNC via simultaneous interactions with the SRP NG-domain and with the ribosome, enforcing a kinetically stable RNC-SRP-SR complex that could minimize RNC loss at the ER membrane (Fig. 2F).

GTP hydrolysis accelerates RNC release

The results above were obtained with GMPPNP to mimic the targeting complex before GTP hydrolysis. We asked whether GTP hydrolysis, which occurs quickly in an activated SRP-SR complex on the RNC (0.03 to 0.25 s⁻¹) (28) and drives disassembly of the SRP-SR NG-dimer (15, 20), removes the stabilizing effect of SR on SRP-RNC association. In support of this model, replacement of GMPPNP with GTP allowed faster dissociation of SRP from the RNC-SRP-SR complex (Fig. 3A, orange, and fig. S5). The dissociation kinetics consisted of a slow phase (56%), which has a rate constant (0.0019 s⁻¹) similar to that observed with the complex formed in GMPPNP and is therefore assigned to the GTP-bound, prehydrolysis RNC-SRP-SR complex, and a fast phase (44%) with a rate constant (∼0.01 s⁻¹) similar to that without SR (Fig. 3, A and D, fig. S5, and table S2), which is attributed to the RNC-SRP complex that was generated after GTP hydrolysis and the rapid SRP-SR disassembly that follows hydrolysis (15, 20). In support of this interpretation, a kinetic simulation based on the previously measured rates of SRP-SR binding and GTP hydrolysis (28) found that the prehydrolysis RNC-SRP-SR complex and RNC-SRP complexes generated following GTP hydrolysis are the dominant species at steady state upon complex assembly in GTP and accumulate to similar concentrations before the addition of chase (fig. S6). These data suggest that GTP hydrolysis can accelerate the dissociation of SRP from the RNC.

Fig. 3. GTP hydrolysis accelerates SRP dissociation from RNC.

(A and B) Kinetics of SRP-RNC dissociation, measured as in Fig. 2 (A and B), in the presence of wild-type SR (A) or the GTPase-deficient mutant SR(R458A) (B) for complexes assembled in GTP or GMPPNP. The data without SR (blue) are the same as that in Fig. 2B and shown for comparison. Fluorescence time traces were normalized as in Fig. 2B except for the reaction with SR(R458A) plus GMPPNP, for which the fluorescence change is too small to quantify. The fluorescence trace for this reaction was therefore normalized to the intensity at time 0. (C) Kinetics of SRP-RNC dissociation with the hyperactive GTPase mutant SRP_Hyper, with or without SR in GTP. (D) Summary of the half-time of SRP-RNC dissociation with different SRP and SR constructs and with GTP or GMPPNP. (E and F) Kinetics of signal sequence release from SRP was measured using the SRP^M-Cy3B-RNC^ATTO647N FRET pair and TKRM as chase. Reactions were carried out with wild-type SRP (E) or SRP_Hyper (F) in the absence and presence of SR with indicated nucleotides. The bottom panels summarize the reaction half-times. (G) The GTPase-deficient SR(R458A) has a translocation defect that is not rescued by increasing SR concentration. Top: Representative autoradiograph of pPL targeting and translocation in the presence of 55 nM SRP, TKRM (0.5 eq/μl), and indicated concentrations of SR(R458A). pPL and PL bands are indicated by arrows. Bottom: Quantification of translocation efficiency based on cleavage of the signal sequence. Data are shown as means ± SD, with n = 3 technical replicates. The lines are fits of the data to Eq. 3. The data with wild-type SR were from Fig. 2E and were measured in parallel with SR(R458A). (H) Model for the role of GTP hydrolysis in allowing faster SRP release from the RNC.

To further test this model, we used the GTPase-deficient SR(R458A) mutant, which removes an arginine finger in the GTPase active site to impair reciprocal GTPase activation in the SRP-SR complex but still allows efficient SRP-SR complex formation (table S1) (28). In the presence of GTP, SRP dissociated more slowly from RNC-SRP-SR(R458A) than from the wild-type targeting complex (Fig. 3, B, green, and D; fig. S5; and table S2). When mutant SR(R458A) was combined with GMPPNP to completely block GTP hydrolysis, SRP dissociation was too slow to be reliably measured (Fig. 3, B, inset, red, and D; fig. S5; and table S2). We further tested an established mutant SRP that hyperactivates GTP hydrolysis (SRP_Hyper; table S1) in the RNC-SRP-SR complex (19), which is predicted to increase the population of the posthydrolysis state in the targeting complex assembled in GTP. Without SR, SRP_Hyper dissociated from RNC with kinetics within twofold of that of wild-type SRP (Fig. 3, C and D, fig. S5, and table S2). In the presence of GTP and SR, the dissociation of SRP_Hyper from RNC occurred at the same rate as that without SR (Fig. 3, C and D, fig. S5, and table S2). Thus, the stabilizing effect of SR on RNC-SRP association anticorrelated with the accumulation of SRP-SR complex in the posthydrolysis state.

To test whether GTP hydrolysis played a similar role during RNC handover at the ER, we repeated the pulse-chase measurements using TKRM instead of excess unlabeled SRP as the chase. Previous studies showed that incubation of RNC_pPL translated in rabbit reticulocyte lysate (RRL) with ER microsomes induces the loss of crosslink of a Bpa probe at residue 12 in the pPL nascent chain to SRP54, with concurrent acquisition of a new cross-link to Sec61α and TRAM, indicating successful handover of the signal sequence upon its release from SRP (32–34). In agreement with these earlier observations, TKRM at concentrations sufficient for cotranslational pPL translocation (Fig. 2E) led to the loss of FRET between SRP^M-Cy3B and RNC^Atto647N in the RNC-SRP complex (Fig. 3E, fig. S7, and table S2), indicating that a component at the ER membrane induced signal sequence release from the SRP M-domain. Analogous to in-solution SRP-RNC dissociation measurements, conditions that allow SRP-SR assembly but delay GTP hydrolysis, such as GMPPNP and/or the GTPase-deficient SR(R458A) mutant, delayed signal sequence release from SRP (Fig. 3E, green and red, and fig. S7). The stabilizing effect of SR was reversed when the RNC-SRP-SR complex was assembled with GTP to allow hydrolysis (Fig. 3E, orange) and eliminated when the complex was assembled with SRP_Hyper and GTP (Fig. 3F). These results support a role of GTP hydrolysis in facilitating the release of signal sequence from SRP at the ER membrane.

Curiously, under conditions where GTP hydrolysis was blocked using GMPPNP and/or the GTPase-deficient SR(R458A) mutant, the rate of TKRM-induced signal sequence release was faster than that of RNC-SRP dissociation measured in solution using excess SRP as chase (compare Fig. 3E with A and B). This could be due to the presence of ER component(s) that promote signal sequence release from SRP, as recently reported (21). Alternatively or in addition, the signal sequence may be released from SRP without complete dissociation of the RNC-SRP-SR complex at the ER, as described later (Fig. 4).

Fig. 4. ER microsome promotes the prehandover conformation with substrate specificity.

(A) Scheme of diffusion-based smFRET measurements using μsALEX-FAMS. Fluorescently labeled SRPs diffusing through a femtoliter-scale confocal volume are alternately excited with donor and acceptor excitation lasers. Fluorescence emission from both donor (green) and acceptor (red) dyes were measured to calculate donor-acceptor stoichiometry (S) and apparent FRET efficiency (E*) to generate 2D E*-S plots. Doubly labeled particles with both donor and acceptor dyes (S ∼ 0.5) were optically purified, and 1D projection of their data onto the E* axis generates the FRET histogram. (B) smFRET histograms of SRP^Dist probes in the presence and absence of SR, RNC, and microsomes. N is the number of bursts used to construct each histogram. The data were globally fitted to the sum (solid line) of three Gaussian functions corresponding to low1, low2, and high FRET efficiency (dashed lines). Vertical dashed lines indicate the mean FRET efficiency of each state. (C) Summary of high FRET population detected with SRP^Dist for the indicated complexes. Colors denote the different complexes.

GTP hydrolysis in the SRP-SR complex was not thought to be required for protein translocation per se (18). Our findings prompted us to reexamine this classic model. We carried out cotranslational protein-targeting reactions under single-turnover conditions, where the concentration of SRP (55 nM) far exceeded that of the pPL being synthesized (≪20 nM; fig. S8, A and B), such that SRP recycling was not required for the observed targeting and translocation. Because free SRP binds SR 10²- to 10³-fold more weakly and more slowly than RNC-bound SRP (28), the excess free SRP was not expected to effectively compete with RNC-bound SRP for SR binding sites. SR(R458A) exhibited a substantially reduced translocation efficiency compared to wild-type SR in these reactions, even at saturating SR concentrations where bimolecular SRP-SR and SR-ribosome interactions are no longer limiting (Fig. 3G and table S3). These results provide evidence that GTP hydrolysis, which facilitates RNC release from SRP at the ER membrane, is important for efficient protein translocation (Fig. 3H).

An ER component selectively promotes the prehandover conformation

We previously showed that SRαβΔTM induces RNC-bound SRP to sample the prehandover conformation, by measuring intra-SRP FRET between a donor dye (Cy3B) labeled at residue 47 in the SRP54 NG-domain and an acceptor dye (Atto647N) labeled at SRP68 (SRP^Dist; fig. S1A). Docking of the SRP-SR NG-dimer at SRP68/72 brings this dye pair into proximity, reducing their distance from ∼110 Å in the RNC-SRP complex to ∼40 Å in the prehandover conformation (fig. S1A). Microsecond-resolution alternating laser excitation (μsALEX) spectroscopy, which measures diffusing SRP^Dist particles in solution at single-molecule resolution (Fig. 4A), further enables the sensitive and specific detection of the prehandover conformation as a distinct population with high mean FRET efficiency (E* ∼ 0.7; Fig. 4B, rows 1 and 2) (13). However, the prehandover state was similarly populated regardless of whether the ribosome harbors the pPL nascent chain (∼34%) or a signalless sequence (SKAVAARAAGSTVRQR) derived from the cytosolic domain of Sec61β (RNC_Cyto, ∼36%) (Fig. 4, B, row 2, and C, yellow bars), raising questions about how non-ER proteins are rejected.

To probe whether and how components at the ER regulate the rearrangement of SRP to the prehandover conformation, we assembled the RNC-SRP^Dist-SR complex in GMPPNP and incubated it with TKRM. TKRM increased the prehandover population on RNC_pPL to ∼42% but reduced the prehandover population on RNC_Cyto to ∼13% (Fig. 4, B, row 3, and C, blue bars). Similar effects of the ER membrane were observed when RNC-SRP^Dist was presented to salt-washed microsome (KRM) to assemble a targeting complex with the endogenous SR at the ER membrane, except that the prehandover conformation was even more populated, reaching ∼50% with RNC_pPL and ∼20% with RNC_Cyto (Fig. 4, B, row 4, and C, green bars). The stabilizing effects of TKRM and KRM on the prehandover complex persisted when the pPL nascent chain elongated to 171 amino acids (Fig. 4, B and C). On the other hand, the prehandover conformation was not observed in the absence of RNC or SR even after prolonged incubation with microsome (Fig. 4B, column 1 and row 1), excluding the possibility that our observations stemmed from free SRP that had been completely displaced from the RNC by the microsome. These results show that a component on the ER membrane promotes the prehandover conformation in a signal sequence–selective manner.

We further noted that, for the RNC-SRP-SR complexes formed on TKRM and KRM, the prehandover conformation could be observed for up to 60 min after the complex was assembled (fig. S9A). In comparison, signal sequence release from the SRP M-domain occurred at a much faster rate, with a half-time of ∼330 s when the RNC-SRP-SRαβΔTM complex in GMPPNP was chased by TKRM (Fig. 3D and table S2) and ∼155 s when RNC-SRP was chased by KRM (fig. S9B and table S2). In addition, incubation of the SRP^Dist-RNC complex with KRM in GMPPNP led to efficient cosedimentation of SRP^Dist with the microsome (fig. S9C), indicating that SRP^Dist was stably associated with the ER under conditions of the μsALEX measurement. These observations imply that, before GTP hydrolysis, a partial handover intermediate could accumulate in which the pPL signal sequence was displaced from the SRP M-domain, while SRP-SR remains bound to the RNC on TKRM or KRM. Collectively, the results in this section show that a component at the ER membrane selectively promotes the prehandover conformation of the targeting complex for signal sequence bearing RNCs and raises the possibility of stepwise RNC transfer at the ER membrane.

Accurate timing of GTP hydrolysis ensures efficient targeting

In the prehandover conformation, the C-terminal tail of SRP72 and G232 of 7SL insert into the composite active site in the SRP-SR NG-dimer (19). Disruption of these interactions in SRP_Hyper led to faster GTP hydrolysis (19), indicating that these interactions in the prehandover conformation delay GTP hydrolysis. This model is further supported by the anticorrelation between GTPase activity and the population of the targeting complex in the prehandover state (Fig. 5A and fig. S10A) (13). However, the role of delayed GTP hydrolysis in the prehandover complex has remained unclear.

Fig. 5. Fine-tuned GTPase activity in the SRP-SR complex is required for optimal protein targeting.

(A) GTPase rate of the SRP-SR complex (k_cat) correlates inversely with formation of the prehandover conformation. The prehandover population in RNC_pPL-SRP_Hyper-SR and 80S-SRP-SR were measured herein (fig. S10, A and B). All other values were reported previously (13, 19, 28). (B and C) SRP- or SRP_Hyper-mediated pPL targeting to KRM (B) or to SRαβΔTM and TKRM (C). Top: Representative autoradiograph. Bottom: Quantification of translocation efficiency. The reactions in (C) used 100 nM SRP or SRP_Hyper. Different symbols (● and ▵) depict data from two technical replicates. The data at 3 nM in (C) were from the maximal translocation efficiency in fig. S8F. Lines are global fits of all the replicate data to Eq. 3. (D) Top: Signal sequence variants tested. Bottom: Translocation efficiency of indicated substrates by wild-type SRP/SR (blue), SRP plus SR(R458A) (hypo-GTPase; red), or SRP_Hyper plus SR (hyper-GTPase; green). Reactions contained 60 nM SRP, 250 nM SRαβΔTM, and TKRM (0.5 eq/μl), all at saturating concentrations. The data were quantified from fig. S8 and shown as means ± SD, with n ≥ 3 technical replicates. SRP- and SR-independent translocation was subtracted. The data for the three substrates with optimal signal sequences were combined to improve statistics. (E) Model to explain the translocation defects of hyper- and hypo-GTPase mutants. Vertical arrows show GTP hydrolysis, and curved arrows show RNC release/handover, with line thickness indicating relative rates. With wild-type SRP and SR (red), delayed GTP hydrolysis in the prehandover complex provides a time window to allow productive RNC handover following GTP hydrolysis. With GTPase-deficient SR(R458A) (green), RNC release and handover occur more slowly. With SRP_Hyper (blue), premature GTP hydrolysis uncouples RNC release from productive handover.

To address this question, we compared wild-type SRP and SRP_Hyper in cotranslational pPL targeting. In reactions using purified SRP and KRM, which contains endogenous SR, SRP_Hyper reduced pPL translocation to undetectable levels (Fig. 5B). This was unexpected, as SRP_Hyper could mediate translocation reactions reconstituted using purified SRP, SRαβΔTM, and TKRM, albeit less efficiently than wild-type SRP (Fig. 5C). Independent smFRET measurements showed that the prehandover conformation was only 5% less abundant with RNC-SRP_Hyper than with wild-type RNC-SRP on KRM (fig. S10, B and C, green), indicating that the near complete loss of translocation with SRP_Hyper cannot be attributed to its failure to attain the prehandover conformation. Our previous GTPase measurements also excluded a defect of SRP_Hyper in efficient assembly with SR (19). We first hypothesized that the endogenous SR concentration on KRM is too low to support efficient targeting with SRP_Hyper, as close inspection of the SR concentration dependences of the translocation reaction with SRαβΔTM and TKRM suggested that the defect of SRP_Hyper was stronger at lower SR concentrations (Fig. 5C). Quantitative Western blot analysis gave an estimated concentration of ∼3 nM endogenous SR on KRM in the targeting reaction (fig. S8, C to E), likely below the concentration required for efficient translocation even in the reaction with SRαβΔTM and TKRM (Fig. 5C, arrow). To test this hypothesis, we repeated the translocation reaction using 3 nM SRαβΔTM and TKRM. In agreement with this hypothesis, translocation with wild-type SRP was significantly reduced, and SRP_Hyper showed a larger translocation defect compared to wild-type SRP under these conditions (fig. S8F). Thus, the translocation defect of SRP_Hyper was due in part to the fact that SRP_Hyper-SR association at endogenous SR concentrations was outcompeted by rapid GTP hydrolysis in the SRP_Hyper-SR complex, which drives their dissociation (Fig. 5E, vertical blue arrow). As the rate of SRP_Hyper-SR association is concentration dependent, higher SR concentrations allowed more SRP_Hyper-SR complexes to accumulate at steady state and thus rescued translocation. On the other hand, translocation by wild-type SRP was less efficient, whereas translocation by SRP_Hyper was more efficient, with 3 nM SRαβΔTM plus TKRM than with equivalent concentration of native SR on KRM (Fig. 5B versus fig. S8F). This implies that the ER microsomal environment imposes a stricter requirement for regulated GTP hydrolysis compared to SRαβΔTM on TKRM, which further contributed to the severe translocation defect of SRP_Hyper.

Last, in the targeting reaction reconstituted with SRP_Hyper, SRαβΔTM, and TKRM, the translocation efficiency of pPL was ∼20% lower than that with wild-type SRP even at saturating concentrations of SRP and SRαβΔTM (Fig. 5C), indicating an additional defect of SRP_Hyper in a unimolecular step after the RNC-SRP_Hyper-SR complex has assembled. This is puzzling, as TKRM-induced signal sequence release from SRP occurred facilely with the RNC-SRP_Hyper-SR complex (Fig. 3F), and suggests that not all signal sequences released from SRP_Hyper are successfully handed over for translocation. In the simplest model, this defect could stem from faster GTP hydrolysis in the RNC-SRP_Hyper-SR complex, which gives the complex a much shorter time for productive RNC handover. An additional source of the defect was suggested by smFRET measurements using the SRP^Dist probe for RNC-SRP-SRαβΔTM complexes docked on TKRM, which showed that formation of the prehandover conformation was less efficient with SRP_Hyper than with wild-type SRP (fig. S10, B and C). Regardless of the precise mechanism, these data indicate that the failure to regulate GTP hydrolysis in the prehandover complex leads to the decoupling of signal sequence release from productive cargo handover.

The reduced translocation efficiency observed with both SRP_Hyper and the GTPase-deficient SR(R458A) mutant suggested that a fine-tuned GTPase rate in the targeting complex optimizes targeting efficiency. To address whether regulated GTP hydrolysis differentially affects diverse substrates proteins, we tested variants of pPL in which the hydrophobicity of the signal sequence is varied by systematically altering Ala/Leu ratios (Fig. 5D) (35). pPL variants with synthetic signal sequences 2A8L and 3A7L translocated with efficiencies similar to that of wild-type pPL (fig. S11) and were therefore grouped as “optimal” substrates (Fig. 5D). The translocation of 4A6L was moderately reduced compared to the optimal substrates, whereas 5A5L reduced translocation to near background levels (fig. S11, “ctrl”). We further tested a model substrate containing residues 1 to 71 of the β₁-adrenergic receptor (β₁AR-TM1), which contains a TMD with type I topology and was inserted by the EMC instead of Sec61p complex (Fig. 5D) (36). β₁AR-TM1 was fused to part of the disordered cytosolic domain of Sec61β (residues 2 to 68) to increase solubility, and five engineered methionines were inserted at the C terminus to increase radioactive signal for its detection. Both SRP_Hyper and SR(R458A) reduced translocation efficiency across all the pPL variants and β₁AR-TM1 (Fig. 5D and fig. S11). Thus, precisely timed GTP hydrolysis is required for the optimal targeting and translocation of a diverse array of SRP substrate proteins with distinct physicochemical properties.

DISCUSSION

The handover of translating ribosomes from SRP to translocation machineries at the ER is required for the initiation of protein translocation and the entry of over 30% of the nascent proteome to the endomembrane system in eukaryotic cells. Despite its importance, the mechanism of this cargo handover event has remained elusive because of its transient nature. In this work, single-molecule and transient kinetic measurements revealed intricate regulations of RNC handover by the SR, GTP hydrolysis, and the ER membrane. Our results provide a molecular model for coordinated cargo handover during cotranslational protein targeting and translocation and revealed previously unidentified roles of GTP hydrolysis and the ER membrane in the SRP pathway.

Our work revealed dual roles of the SR in regulating the interaction of SRP with the translating ribosome to shape cargo release. First, SR dissolves two key interactions that characterize the initial RNC-SRP complex: the contact of the SRP NG-domain with uL23/29 on the ribosome (13, 19) and the docking of the signal sequence at the SRP M-domain (Figs. 1, D to E, and 6, step 2). Elongation of the nascent chain enforces the SR-induced release of the signal sequence and allows it to move further away from the M-domain and the ribosome (Figs. 1, D to E, and 6, step 4). These SR-induced changes free up the ribosome exit site and the signal sequence, enabling their search for and subsequent interaction with the Sec61p machinery. On the other hand, SR stabilizes the association of SRP with the RNC before GTP hydrolysis (Fig. 2). Our mutational analyses suggest that this stabilization arises, at least in part, from the ability of SR to simultaneously bind with SRP via NG-dimerization and with the ribosome via its K/R-rich linker, thus acting as a tether between the two (Fig. 2). These dual roles of SR prime the ribosome and signal sequence for handover to translocation machineries, while also preventing the premature loss of RNC that could abort targeting.

Fig. 6. Model for regulated cargo handover during cotranslational protein targeting and translocation.

Step 1: SRP recognizes RNC bearing an ER signal sequence, which induces SRP into the proximal conformation. Step 2: Initial SRP-SR association via GTP-dependent dimerization of their NG-domains. This induces the detachment of the NG-dimer from uL23/29 on the ribosome and the undocking of signal sequence from the SRP M-domain. Step 3: The ER membrane selectively induces the targeting complex to the prehandover conformation for signal sequence–bearing RNCs but not for signalless ribosomes (purple arrow). Step 4: Elongation of the nascent chain may allow the signal sequence to initiate interaction with Sec61p at the ER. Step 5: Regulated GTP hydrolysis from the prehandover complex severs the stable SRP-SR NG-dimer. Step 6: Dissociation of SRP from the posthydrolysis complex frees the ribosome for handover to the translocase. “?” denotes additional unknown steps that could be involved in completing the RNC transfer. Step 7: Nucleotide exchange and recycling of SRP.

However, this SR-induced stabilization would impede the dissociation of SRP from the ribosome, which is required for subsequent formation of the RNC-Sec61p translocation complex (Fig. 6, step 6; PDB: 3J7R) (37). Our results suggest that this barrier is overcome in part by GTP hydrolysis, which drives dissociation of the stable SRP-SR NG-dimer. This model is supported by the observation that conditions that increase the population of the posthydrolysis SRP-SR complex led to faster SRP dissociation from the RNC, whereas conditions that increase the population of the prehydrolysis complex had the opposite effect (Fig. 3). Moreover, mutant SR(R458A), which impairs GTPase activation (28), reduced the efficiency of cotranslational protein targeting even under conditions where the recycling of SRP and SR were not required (Fig. 3G). These results suggest that GTP hydrolysis initiates more facile release of SRP from the RNC, providing the first evidence for a direct role of GTP hydrolysis in regulating cotranslational protein targeting and translocation.

Our data further suggest that the precise timing of GTP hydrolysis plays a critical role in the SRP pathway. In the prehandover conformation, evolutionarily conserved interactions between SRP72/SRP RNA and the composite GTPase active site lead to delayed GTP hydrolysis (13). Removal of these regulatory interactions in mutant SRP_Hyper, which accelerates GTP hydrolysis ∼5-fold (19), impaired cotranslational protein targeting and translocation via two mechanisms (Fig. 5B). At endogenous concentrations of SR, such as those found on the canine pancreatic KRM, SRP-SR association is unable to compete with rapid GTP hydrolysis by SRP_Hyper that drives their dissociation (Fig. 5E, vertical blue arrow). Even when this association defect is overcome by increased SR concentration in reactions using soluble SRαβΔTM and TKRM, signal sequence release from SRP_Hyper is less effectively coupled to cargo handover (Fig. 5E, curved blue arrow). Thus, delayed GTP hydrolysis in the prehandover complex is critical for establishing a substantial population of handover-competent targeting complexes at endogenous SR concentrations at the ER membrane. The native microsome further imposes an even stricter requirement for delayed GTP hydrolysis compared to SRαβΔTM reconstituted on TKRM, suggesting an additional layer of regulation that remains to be understood.

Last, the ER membrane promotes the formation of the prehandover conformation, which is critical for bringing the ribosome close to the membrane and for regulating GTP hydrolysis. This further emphasizes the importance of this conformational state and suggests an additional mechanism at the ER to enhance the fidelity of protein targeting, in which signal sequence–containing ribosomes more productively transition to translocation whereas signalless ribosomes are rejected. For targeting complexes assembled in GMPPNP on the ER microsome, signal sequence release from SRP occurred within 3 min (Fig. 3E), whereas the prehandover complex persisted for an hour (Fig. 4 and fig. S9). These observations suggest that, before GTP hydrolysis, an intermediate can accumulate in which the signal sequence is released from SRP and can search for or engage with translocation machineries, while the ribosome remains bound to SRP and SR, providing a concert pathway for the transition to the translocation complex.

Although the ER factor that exerts this conformational regulation is unknown, the observation that this effect persists in trypsin-digested TKRM indicates that it is deeply embedded in the ER membrane and protected from proteolysis. In the simplest model, the engagement of signal sequence with the Sec61p complex could spatially confine SRP and SR between the ribosome and ER membrane to favor formation of the prehandover state. Additional factors that contribute to this regulation include TRAP (38), TRAM (39), TMEM208 (21), and the TMD of SRβ (30). Further studies will be required to identify the factor(s) underlying this regulation.

Our findings, together with previous work (11, 13, 27, 28), provide a high-resolution molecular model for the membrane-associated events in the SRP pathway (Fig. 6). Recognition of signal sequence–bearing RNCs (step 1) activates SRP to efficiently assemble with SR (step 2). GTP-dependent assembly of the SRP-SR NG-dimer induces this dimer to detach from the ribosome exit site and the signal sequence to undock from the SRP M-domain (step 2), freeing both components on the RNC for subsequent interaction with translocation machineries. At this stage, the association of SRP with the RNC is stabilized by SR, which interacts with SRP via NG-dimerization and with the ribosome via its K/R-rich linker. Docking of the SRP-SR NG-dimer at the distal site of SRP generates the prehandover conformation, which brings the translating ribosome closer to the ER membrane (step 3). Formation of the prehandover complex is promoted by components at the ER in a signal sequence–selective manner, such that signalless ribosomes are more likely rejected (purple arrow). Delayed GTP hydrolysis in the prehandover complex provides a time window during which the nascent chain searches for the appropriate translocation machinery, and this search could be facilitated by elongation of the nascent chain, which further weakens signal sequence docking and increases its diffusion range (step 4). GTP hydrolysis (step 5), which dissociates the SRP-SR NG-dimer, enables more facile release of SRP from the ribosome (step 6). The regulated release of SRP from the prehandover complex is more likely to be coupled to productive RNC handover (step 6) compared to release from alternative conformations, allowing the SRP pathway to successfully transition from targeting to translocation.

Protein-targeting machineries, especially those dedicated to integral membrane proteins, are subject to extensive spatial control: In the aqueous environment, they must bind tightly to substrate proteins to minimize the rapid and irreversible aggregation of the nascent protein. Upon arrival at the target membrane, however, they need to facilely unload the cargo to translocation/insertion machineries to initiate the folding and maturation of substrate proteins. These molecular handovers are ubiquitous across diverse organisms and membrane protein biogenesis pathways, but their underlying mechanism is elusive for many pathways. Our work here provides a paradigm for the intricate molecular regulations that ensure efficient, productive, and selective cargo handover in an essential protein-targeting machine, which could shed light on analogous events in other targeting pathways that are subject to the same constraints.

MATERIALS AND METHODS

Preparation of proteins and complexes

SRP proteins (SRP19, SRP9/14, SRP68/72, and SRP54), SR (SRαβΔTM) (28), and engineered MmPylRS (Methanosarcina mazei pyrrolysyl-tRNA synthetase) (40) were expressed and purified as described. SRP 7SL RNA and PyltRNA were in vitro transcribed by T7 polymerase using a homemade transcription kit and purified by acid phenol extraction and isopropanol precipitation (11). SRP was assembled in vitro from purified subunits and 7SL RNA and purified over a DEAE anion exchange column as described previously (11).

mRNAs for RNC generation and engineered MmPyltRNA for amber suppression were transcribed using T7 polymerase and purified by LiCl precipitation, as described previously (40). To immobilize RNCs on neutravidin-coated surface in the smTIRFM assay, a 4:5 ratio of GTP and 5′-biotin–guanosine 5′-monophosphate (GlpBio) was used during transcription to generate mRNAs with a 5′ biotin label (11). For protein-targeting and translocation assays, the mRNAs for full-length pPL, pPL signal sequence variants, and β₁AR-TM1 were transcribed using the pSPBP4 vector and the SP6 MEGAscript Kit (Ambion) (27).

RNC preparation

To generate RNCs, polymerase chain reaction amplicons encoding, from 5′ to 3′, the encephalomyocarditis virus internal ribosomal entry site, 3xFLAG tag, the HRV 3C protease cleavage site, and the coding sequences for specified N-terminal sequences of different protein substrates were transcribed in vitro using T7 polymerase. RNCs were generated and purified as described (11, 40). Briefly, mRNAs were in vitro translated in RRL for 30 min at 32°C. RNCs were purified by sedimentation over a high-salt cushion [50 mM KHepes (pH 7.5), 1 M KOAc, 15 mM Mg(OAc)₂, 0.5 M sucrose, 0.1% Triton X-100, and 2 mM dithiothreitol (DTT)] and immunopurification with anti-FLAG agarose (Pierce; 0.03 vol of translation reaction). After cleavage of the FLAG tag by the HRV 3C protease, immunopurified RNCs were sedimented over a high-salt cushion again and resuspended in SRP assay buffer [50 mM KHepes (pH 7.5), 150 mM KOAc, 5 mM Mg(OAc)₂, 10% glycerol, 2 mM DTT, and 0.04% Nikkol (octaethylene glycol monododecyl ether or C12E8)] (40). To fluorescently label the RNC, mRNAs encoding pPL nascent chains with amber codon at R12 were translated in the presence of TCOK (axial-trans-cyclooct-2-en-l-lysine), MmPyltRNA, and MmPylRS. For TIRFM assays, RNCs were translated using 5′-biotin–labeled mRNA for immobilization on the coverslip surface.

Microsome preparation

KRM and TKRM were prepared using an RM preparation from canine pancreas (a gift from H. Bernstein) following published protocols (41). TKRM were stored at 2 equivalent (eq)/μl [or 100 A₂₈₀ (absorbance at 280 nm) U/ml] concentration in 50 mM KHepes (pH 7.5), 250 mM sucrose, and 1 mM DTT (41).

Biochemical measurements

Cotranslational protein targeting and translocation

Reactions were carried out as described previously (27, 31). Briefly, 0.5 μg of mRNA encoding indicated that substrate proteins were translated in SRP-free wheat germ lysate containing [³⁵S]-methionine for 2 min, followed by addition of purified SRP, SRαβΔTM and TKRM, or SRP and KRM at indicated concentrations. GTP provides energy for translation. Reactions were incubated at 25°C for 40 min and quenched with 2× SDS sample buffer. Untranslocated and translocated proteins were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and detected by autoradiography. For pPL and signal sequence variants of pPL, the molecular weight shift from 26 kDa (pPL) to 22 kDa (PL) after signal sequence cleavage was monitored. Translocation efficiency was calculated using Eq. 1

$$\text{Fraction translocated}=\frac{\frac{8}{7}\times \text{PL}}{\frac{8}{7}\times \text{PL}+\text{pPL}}$$ (1)

in which PL and pPL are the autoradiography signals for the respective protein bands and 8/7 corrects for the different number of methionine residues in pPL compared to PL. For β₁AR-TM1, the molecular weight shift from 16 to 22 kDa after its glycosylation was monitored. Translocation efficiency was calculated using Eq. 2

$$\text{Fraction translocated}=\frac{\text{Gly}-\mathrm{\beta }}{\text{Gly}-\mathrm{\beta }+\mathrm{\beta }}$$ (2)

in which Gly-β and β are the autoradiography signals for glycosylated and unglycosylated β₁AR-TM1, respectively. The SR concentration dependences of translocation were fit to Eq. 3

$$\text{Fraction translocated}=\frac{T_{\text{max}}\times \left[\text{SR}\right]}{K_{\mathrm{m}}+\left[\text{SR}\right]}+T_0$$ (3)

where T_max is the translocation efficiency at saturating SR concentrations, K_m is the SR concentration required to obtain 50% of T_max, and T₀ is translocation efficiency in the absence of SRP and SR. These parameters were obtained by fitting experiments in which SRP concentration was held constant, while SR was titrated. When SRP was titrated instead (Fig. 5B), the [SR] term in Eq. 3 was replaced with [SRP].

SR cosedimentation with ribosome

Cosedimentation assays were carried out as described in (27). Briefly, 400 nM SRαβΔTM (wild-type or mutant) were incubated with 150 nM 80S in 70 μl of reactions in 50 mM KHepes (pH 7.5), 100 mM KOAc, 5 mM Mg(OAc)₂, 0.04% Nikkol, and 2 mM DTT at 25°C for 10 min. The mixture was loaded onto a 110-μl sucrose cushion (1 M) in the same buffer and ultracentrifuged at 100,000 rpm for 2 hours at 4°C in a TLA100 rotor (Beckman Coulter). The pellet fractions were resuspended in 20 μl of 1× SDS loading buffer. Equal volumes of the total and pellet samples were analyzed on SDS-PAGE and Coomassie blue staining and scanned on a LI-COR Odyssey imager using a wavelength of 700 nm.

SRP cosedimentation with microsome

A total of 80 nM SRP^Dist, with or without 200 nM RNC_pPL91, and 0.34 eq of KRM were incubated in 50 mM KHepes (pH 7.5), 5 mM Mg(OAc)₂, 100 μM GMPPNP, 0.04% Nikkol, and 2 mM DTT at 25°C for 0.5 to 30 min. The volume of purified SRP stock (which contains 600 mM KOAc) was adjusted to reach a final KOAc concentration of 100 mM. The mixture was then loaded onto a 40-μl sucrose cushion (0.5 M) in the same buffer and ultracentrifuged at 55,000 rpm at 4°C for 3 min in a TLA100 rotor (Beckman Coulter). The pellet fractions were resuspended in 60 μl of 1× SDS loading buffer. Equal amounts of the total, supernatant, and pellet samples were analyzed on SDS-PAGE gels. The gels were scanned on a Typhoon FLA 9000 imager to detect the fluorescence signal of ATTO647N on SRP68 using the 625-nm channel.

Quantitative Western blot analysis of SR on microsome

To estimate the concentration of endogenous SR in the microsome, serial dilutions of KRM and purified SRαβΔTM were ran on the same SDS-PAGE gel and analyzed by immunoblotting using anti-SRα antibody (Bio-Techne, H00006734-B02P). The SRαβΔTM concentration dependence of the SRα signal was used to construct a standard curve to determine the concentration of SRα on KRM samples.

Quantification of pPL generated during in vitro translation-translocation

RNC_pPL91 was generated in RRL in the presence of ³⁵S-methionine and partially purified by sedimentation over a high-salt sucrose cushion as described above. The ribosome concentration of the RNC sample was determined using A₂₆₀ and an extinction coefficient of 20 nM per A₂₆₀. Wheat germ translations of pPL in the presence of ³⁵S-methionine using different mRNA concentrations were analyzed on the same autoradiograph as serial dilutions of partially purified ³⁵S-labeled RNC_pPL91.

Fluorescence labeling

Nascent chains on the RNC were labeled via amber suppression reactions to incorporate the TCOK unnatural amino acid at an engineered amber codon in the mRNA, followed by click reactions with tetrazine-conjugated ATTO647N, as described before (11, 40). Purified SRP54(K47C) (NG-domain) and SRP54(G363C) (M-domain), based on a cyslite SRP54, were labeled with Cy3B using thiol-maleimide chemistry as described (11, 13, 28). Purified SRP68/72 with a ybbR tag inserted at P149 was labeled with CoA-ATTO647N using sfp-mediated conjugation (13). Cy3B-labeled SRP54(G363C) was assembled with other SRP protein and RNA subunits to generate SRP^M-Cy3B, as described in (11). Cy3B-labeled SRP(K47C) and ATTO647N-labeled SRP68/72 were assembled with other SRP protein and RNA subunits to generate SRP^Dist, as described in (13).

Ensemble fluorescence measurements

All measurements were conducted in SRP assay buffer [50 mM KHepes (pH 7.5), 100 mM KOAc, 5 mM Mg(OAc)₂, 0.04% Nikkol, and 1 mM DTT] supplemented with bovine serum albumin (BSA; 1 mg/ml) at 25°C on a Fluorolog 3-22 spectrophotometer (HORIBA).

Fluorescence emission spectra were collected using an excitation wavelength of 558 nm and emission wavelengths from 568 to 688 nm. To measure the dissociation of SRP from the RNC-SRP (or RNC-SRP-SR) complex, 150 μl of 4 nM SRP^M-Cy3B, 20 nM ${\text{RNC}}_{\text{pPL}91}^{\text{ATTO}647\mathrm{N}}$ (and 400 nM SR where indicated) were preincubated for 5 to 10 min in 100 μM GTP or GMPPNP. A total of 150 μl of unlabeled SRP (200 nM; or 200 nM SRP preincubated with 400 nM SR in 100 μM GTP or GMPPNP) was then added to initiate dissociation of the RNC-SRP or RNC-SRP-SR complex, respectively. To measure microsome-driven signal sequence release from SRP, 150 μl of KRM (0.08 eq/μl; or TKRM preincubated with 400 nM SRαβΔTM) was added to a preformed ${\text{RNC}}_{\text{pPL}91}^{\text{ATTO}647\mathrm{N}}$-SRP^M-Cy3B complex (or ${\text{RNC}}_{\text{pPL}91}^{\text{ATTO}647\mathrm{N}}$-SRP^M-Cy3B-SRαβΔTM complex) as chase. The fluorescence recovery of SRP^M-Cy3B was recorded over time. Biphasic time traces were fit to Eq. 4

$$F=F_{\text{max}}-\mathrm{\Delta }{F}_{\text{fast}} e^{-k_{\text{fast}}}-\mathrm{\Delta }{F}_{\text{slow}} e^{-k_{\text{slow}}}$$ (4)

in which F_max is the fluorescence intensity when the reaction reached the new equilibrium, k_fast and k_slow are the rate constants of the fast and slow phases, respectively, and ΔF_fast and ΔF_slow are the magnitude of fluorescence changes in the fast and slow phases, respectively. Fluorescence time traces are normalized such that the fluorescence signal is 0 at t = 0 and 1 when the reaction reached equilibrium (i.e., F_max = 1). Single exponential time traces were fit to Eq. 5

$$F=F_{\text{max}}-\mathrm{\Delta }F\times e^{-\mathit{kt}}$$ (5)

in which k is the SRP dissociation constant and F_max and ΔF are as defined in Eq. 4. Fluorescence time traces were normalized such that F_max = 1 and ΔF = 1. All the fitting results are shown in table S2, and the half-times of the SRP-RNC dissociation are summarized in Fig. 3D.

To obtain the equilibrium K_d of SRP and RNC bearing the wild-type or the 2R mutant signal sequences of pPL, 1 nM SRP^M-Cy3B and 2 nM ${\text{RNC}}_{\text{pPL}91}^{\text{ATTO}647\mathrm{N}}$ were incubated for 10 min at room temperature, followed by the addition of 2, 4, 8, 16, 32, or 64 nM unlabeled RNC_pPL91 with wild-type or 2R mutant signal sequences. The mixtures were incubated at 4°C overnight, and the recovery of SRP^M-Cy3B fluorescence signals due to dissociation from ${\text{RNC}}_{\text{pPL}91}^{\text{ATTO}647\mathrm{N}}$ was recorded at 576 nm. Observed FRET efficiencies were plotted as a function of unlabeled RNC concentrations and fit to Eq. 6

$$\begin{array}{c}E=E_{\text{SRP}}·\frac{\left[\text{SR}{\mathrm{P}}^{\ast }\text{RN}{\mathrm{C}}^{\ast }\right]}{\left[\text{SR}{\mathrm{P}}^{\ast }\right]}\\ =E_{\text{SRP}}·\frac{-[K^{\ast }(\left[\text{RNC}\right]-\left[\text{SR}{\mathrm{P}}^{\ast }\right])+K(\left[\text{SR}{\mathrm{P}}^{\ast }\right]+\left[\text{RN}{\mathrm{C}}^{\ast }\right])]+\sqrt{{[K^{\ast }(\left[\text{RNC}\right]-\left[\text{SR}{\mathrm{P}}^{\ast }\right])+K(\left[\text{SR}{\mathrm{P}}^{\ast }\right]+\left[\text{RN}{\mathrm{C}}^{\ast }\right])]}^2+4\left[\text{RN}{\mathrm{C}}^{\ast }\right]\left[\text{SR}{\mathrm{P}}^{\ast }\right]K(K^{\ast }-K)}}{2(K^{\ast }-K)}\end{array}$$ (6)

where $E_{\text{SRP}}$ is the FRET efficiency in the absence of unlabeled RNC, $\left[\text{SR}{\mathrm{P}}^{\ast }\right]$ is the total concentration of SRP^M-Cy3B (1 nM), $\left[\text{RNC}\right]$ is the concentration of unlabeled RNC, $\left[{\text{RNC}}^{\ast }\right]$ is the concentration of ${\text{RNC}}_{\text{pPL}91}^{\text{ATTO}647\mathrm{N}}$ (2 nM), K is the dissociation constant of unlabeled RNCs from SRP, and K^* is the dissociation constant of ${\text{RNC}}_{\text{pPL}91}^{\text{ATTO}647\mathrm{N}}$ from SRP, which was previously determined to be 0.14 nM.

Kinetic simulation

Kinetic simulations were performed using the SimBiology toolbox in MATLAB. Because RNC was present in excess and at a concentration far above the K_d for SRP binding, all SRP molecules were assumed to be RNC bound. In addition, SRP-SR dissociation after GTP hydrolysis and guanosine diphosphate–to–GTP exchange on SRP and SR are rapid relative to the timescale of all other reaction steps and were therefore assumed to occur instantaneously. Under these assumptions, the reaction scheme was simplified to the reversible association of the SRP-RNC complex with SR, characterized by the experimentally determined (28) association and dissociation rate constants (k_on and k_off), followed by GTP hydrolysis within the SRP-RNC-SR complex, using the previously measured (28) catalytic rate constant (k_cat; fig. S6A). Initial concentrations of SRP·RNC and SR were set to 2 and 200 nM. The values of k_on, k_off, and k_cat were 0.0014 nM⁻¹ s⁻¹, 0.022 s⁻¹, and 0.15 s⁻¹, respectively. Simulations were run for 300 s, although steady state was reached within ∼20 s.

TIRFM-based smFRET measurements

Surface preparation and sample immobilization

Quartz microscope slides and coverslips were aminosilanized using VECTABOND (Vector Laboratories) and functionalized with biotin–polyethylene glycol (Laysan Bio). Surfaces were passivated for ≥1 hour at 25°C in passivation buffer [5% Tween 20 and BSA (1 mg/ml)] and rinsed with 50 mM KHepes (pH 7.5). NeutrAvidin (Thermo Fisher Scientific) was introduced into each flow channel at a final concentration of 0.5 mg/ml and incubated for 10 min. Excess NeutrAvidin was removed by washing with imaging buffer [SRP assay buffer supplemented with BSA (1 mg/ml) and 100 μM GMPPNP] and an oxygen-scavenging system (4 mM Trolox, 2.5 mM protocatechuic acid, and 50 nM protocatechuate-3,4-dioxygenase). Biotinylated RNCs were diluted to 1.5 nM in imaging buffer and incubated in NeutrAvidin-functionalized flow channels for 10 min at 25°C to allow for surface immobilization. Unbound complexes were removed by washing with imaging buffer. SRP was subsequently introduced at a final concentration of 2 nM.

Single-molecule FRET measurement

Fluorescence imaging was performed using MicroManager software on a custom-built TIRF microscope. ATTO647N-labeled RNCs were identified by excitation at 635 nm. Single-molecule FRET time traces were acquired using donor excitation at 532 nm (Cy3B) in single-excitation mode. Donor and acceptor fluorescence intensities were recorded simultaneously with a temporal resolution of 50 ms.

Single-molecule FRET analysis and kinetic modeling

Donor and acceptor fluorescence channels were aligned and analyzed using iSMS software (42). Fluorescence intensities were corrected for background, donor leakage into the acceptor channel, and differences in donor and acceptor quantum yields and detection efficiencies using a γ correction factor. Apparent FRET efficiencies ($E_{\text{app}}$) were calculated using Eq. 7

$$E_{\text{app}}=\frac{I_{\text{DA}}}{I_{\text{DD}}+I_{\text{DA}}}$$ (7)

in which $I_{\text{DD}}$ and $I_{\text{DA}}$ are donor and acceptor fluorescence intensities, respectively, upon donor excitation. Each data point represents a single FRET event defined by the acquisition time (50 ms). For each experimental condition, individual FRET events from all trajectories were pooled to obtain ≥3000 events (N values are specified in each smFRET histogram), yielding macroscopic equilibrium of FRET efficiency distributions.

FRET trajectories from replicate measurements were extracted from iSMS, truncated at photobleaching events, and pooled for HMM analysis using the hmmlearn Python package, following a population-level analysis framework (43). Models with one to five hidden states were evaluated using Gaussian emission distributions with a full covariance setup, a maximum of 10,000 training iterations, and a convergence tolerance of 10⁻⁵. Start probabilities, transition probabilities, emission means, and emission covariances were optimized (parameters: stmc), and state inference was performed using the Viterbi algorithm. The optimal number of FRET states was determined using the Bayesian information criterion and Akaike information criterion. In all datasets, a three-state model minimized both criteria and was selected to avoid overfitting. Final HMM fitting was performed using three states and 100,000 training iterations (full covariance, convergence tolerance = 10⁻⁵, parameters updated = stmc).

The state-specific mean FRET efficiencies obtained from HMM analysis were used to fit experimental FRET efficiency histograms according to Eq. 8

$$\mathit{\text{PDF}}=\sum _{i=1}^n{A}_i·\frac{1}{{\mathrm{\sigma }}_i\sqrt{2\mathrm{\pi }}}{e}^{-\frac{1}{2}{\left(\frac{E-{\mathrm{\mu }}_i}{{\mathrm{\sigma }}_i}\right)}^2}$$ (8)

in which PDF is probability density function, n is the number of FRET states and set to 3, $A_i$ is the fractional population of the $i$th state, and ${\mathrm{\mu }}_i$ and ${\mathrm{\sigma }}_i$ are the mean and SD of the corresponding Gaussian distribution determined by HMM analysis.

Diffusion-based smFRET measurements

Measurements were performed as described in (13, 44, 45). Fluorescently labeled SRP was diluted to a final concentration of 100 to 200 pM in SRP assay buffer supplemented with BSA (0.1 mg/ml) and the indicated effectors and/or interaction partners. Before measurements, samples were incubated at 25°C for 5 to 10 min to ensure complete complex formation. Measurements were carried out in a sealed chamber assembled by sandwiching a perforated silicone sheet (Grace Bio-Labs) between two coverslips to minimize evaporation during data acquisition. Data were collected over a period of 20 to 60 min using a microsecond alternating laser excitation–fluorescence-aided molecule sorting (μsALEX-FAMS) setup equipped with two single-photon avalanche diodes (PerkinElmer) and continuous-wave 532-nm (CNI Laser) and 638-nm (Opto Engine LLC) excitation sources operating at 150 and 100 μW, respectively.

All diffusion-based smFRET data were analyzed using FRETBursts, an open-source burst analysis toolkit for confocal smFRET (46). A dual-channel burst search (47) was performed to isolate the photon streams from the particles containing FRET pairs, separating them from particles labeled with donor-only or acceptor-only dye. Each burst, assumed to originate from a single fluorescent species, was identified as a minimum of 10 consecutive detected photons with a photon count rate at least sixfold above the background count rate during both donor and acceptor excitation periods. Because the background rate can fluctuate within a measurement, the background rate was computed for every 50-s interval according to the maximum likelihood fitting of the interphoton delay distribution. To avoid oversampling individual molecules and to improve statistical robustness, photon streams from consecutive bursts were merged when the interburst interval was less than 1 ms.

The identified bursts were further selected according to the following criteria: (i) $n_{\text{DD}}+n_{\text{DA}}\ge 15$ and (ii) $n_{\text{AA}}\ge 15$, where $n_{\text{DD}}$ and $n_{\text{DA}}$ are the background-corrected numbers of photons collected from the donor and acceptor channels, respectively, during the donor excitation period, and $n_{\text{AA}}$ is the background-corrected number of photons collected from the acceptor channel during the acceptor excitation. The apparent FRET efficiency (E*) and stoichiometry (S) for each burst were calculated using Eqs. 9 and 10

$$E^{\ast }=\frac{n_{\text{DA}}}{n_{\text{DD}}+n_{\text{DA}}}$$ (9)

$$S=\frac{n_{\text{DA}}+n_{\text{DA}}}{n_{\text{DD}}+n_{\text{DA}}+n_{\text{AA}}}$$ (10)

FRET histograms were obtained by one-dimensional (1D) projection of the 2D E*-S histograms onto the E* axis and were used to monitor SRP conformational changes in response to the indicated effectors and/or interaction partners. To minimize contamination from donor-only or acceptor-only species, only bursts with 0.2 ≤ S ≤ 0.8 were selected from the 2D E*-S histograms when generating FRET histograms. To quantitatively analyze the effect of interaction partners on SRP conformations, data from all replicates were summed, and the histograms were globally fitted to the sum of three Gaussian functions with shared means and SDs for each Gaussian across conditions.

Supplementary Materials

This PDF file includes:

Tables S1 to S3

Figs. S1 to S11