Translocation of proteins across the endoplasmic reticulum III. Signal recognition protein (SRP) causes signal sequence-dependent and site- specific arrest of chain elongation that is released by microsomal membranes

The previously observed (Walter, et al. 1981 J. Cell Biol. 91:545-550) inhibitory effect of SRP selectively on the cell-free translation of mRNA for secretory protein (preprolactin) was shown here to be caused by a signal sequence-induced and site-specific arrest in polypeptide chain elongation. The Mr of the SRP-arrested nascent preprolactin chain was estimated to be 8,000 corresponding to approximately 70 amino acid residues. Because the signal sequence of preprolactin comprises 30 residues and because approximately 40 residues of the nascent chain are buried (protected from protease) in the large ribosomal subunit, we conclude that it is the interaction of SRP with the amino-terminal signal peptide of the nascent chain (emerged from the large ribosomal subunit) that modulates translation and thereby causes an arrest in chain elongation. This arrest is released upon SRP-mediated binding of the elongation-arrested ribosomes to the microsomal membrane, resulting in chain completion and translocation into the microsomal vesicle.     

Direct probing of the interaction between the signal sequence of nascent preprolactin and the signal recognition particle by specific cross-linking

We have studied the interaction between the signal sequence of nascent preprolactin and the signal recognition particle (SRP) during the initial events in protein translocation across the endoplasmic reticulum membrane. A new method of affinity labeling was used, whereby lysine residues, carrying the photoreactive group 4-(3-trifluoromethyldiazirino) benzoic acid in their side chains, are incorporated into a protein by means of modified lysyl-tRNA, and cross-linking to the interacting component is induced by irradiation. SRP interacts through its Mr 54,000 polypeptide component with the signal sequences of nascent preprolactin chains containing about 70 residues, and with decreasing affinity with longer chains as well; it causes inhibition of elongation. Binding of SRP is reversible and requires the nascent chain to be bound to a functional ribosome. SRP cross-linked to the signal sequence still inhibits elongation but does not prevent it completely. We conclude that SRP does not block the exit site of the polypeptide chain on the ribosome. The SRP receptor of the endoplasmic reticulum membrane displaces the signal sequence from SRP and, even if SRP is cross-linked, releases elongation arrest.     

Translocation of nascent secretory proteins across membranes can occur late in translation.

Signal recognition particle (SRP) causes an arrest in the translation of nascent secretory proteins in a wheat germ cell-free system. In order to examine at what point during the synthesis of a secretory protein its translocation across the endoplasmic reticulum (ER) membrane can occur, SRP was used to arrest nascent chain elongation at various times during a synchronous translation, thus allowing the generation of nascent chains of increasing length. It was found that SRP can still bring about an arrest as late as when an average of two-thirds of nascent IgG light chain was completed. Rough microsomes were added to translations blocked with SRP to determine if such relatively long nascent chains could still be translocated across the membrane. It was found that nascent chains which had been arrested by SRP, regardless of their length, could be translocated into rough microsomes. In the case of IgG light chain, translocation levels of 50% were still observed with nascent chains corresponding to as much as 70-75% of the intact preprotein. Similar results were observed for the nascent bovine prolactin precursor. These results demonstrate that the synthesis of secretory proteins can be uncoupled from their translocation, and that fairly large nascent chains are capable of crossing the membrane of the ER post-translationally.     

Translocation of preproinsulin across the endoplasmic reticulum membrane. The relationship between nascent polypeptide size and extent of signal recognition particle-mediated inhibition of protein synthesis.

Signal recognition particle (SRP) induces elongation arrest of nascent presecretory proteins as the signal peptide protrudes from the large ribosomal subunit. To examine the relationship between the size of the precursor and extent of SRP mediated inhibition of polypeptide chain elongation, we performed in vitro translation experiments in the presence of SRP using a series of truncated preproinsulin mRNA molecules. These precursors possessed the same NH2 terminus as native preproinsulin followed by progressively shorter COOH termini. SRP inhibited translation of precursors as short as 64 amino acids in length, however, the extent of inhibition diminished for shorter precursors. This correlated with a reduction in the time required for ribosomes to transit through the mRNA encoding the shortened precursors. By exploiting a chimeric protein comprising the first 71 residues of preproinsulin fused to the bacterial cytoplasmic enzyme chloramphenicol acetyltransferase, we demonstrate that the largest size a nascent chain can reach and still be susceptible to SRP-mediated elongation arrest is approximately 17 kDa. Our data support the model that SRP binding to the signal peptide is a reversible process even in the absence of microsomal membranes, and that SRP can arrest polypeptide chain elongation at multiple stages during translation.     

The signal sequence receptor, unlike the signal recognition particle receptor, is not essential for protein translocation

Detergent extracts of canine pancreas rough microsomal membranes were depleted of either the signal recognition particle receptor (SR), which mediates the signal recognition particle (SRP)-dependent targeting of the ribosome/nascent chain complex to the membrane, or the signal sequence receptor (SSR), which has been proposed to function as a membrane bound receptor for the newly targeted nascent chain and/or as a component of a multi-protein translocation complex responsible for transfer of the nascent chain across the membrane. Depletion of the two components was performed by chromatography of detergent extracts on immunoaffinity supports. Detergent extracts lacking either SR or SSR were reconstituted and assayed for activity with respect to SR dependent elongation arrest release, nascent chain targeting, ribosome binding, secretory precursor translocation, and membrane protein integration. Depletion of SR resulted in the loss of elongation arrest release activity, nascent chain targeting, secretory protein translocation, and membrane protein integration, although ribosome binding was unaffected. Full activity was restored by addition of immunoaffinity purified SR before reconstitution of the detergent extract. Surprisingly, depletion of SSR was without effect on any of the assayed activities, indicating that SSR is either not required for translocation or is one of a family of functionally redundant components.     

Discrete nascent chain lengths are required for the insertion of presecretory proteins into microsomal membranes

Ribosomes synthesizing nascent secretory proteins are targeted to the membrane by the signal recognition particle (SRP), a small ribonucleoprotein that binds to the signal peptide as it emerges from the ribosome. SRP arrests further elongation, causing ribosomes to stack behind the arrested ribosome. Upon interaction of SRP with its receptor on the ER membrane, the translation arrest is released and the ribosome becomes bound to the ER membrane. We have examined the distribution of unattached and membrane-bound ribosomes during the translation of mRNAs encoding two secretory proteins, bovine preprolactin and rat preproinsulin I. We find that the enhancement of ribosome stacking that occurs when SRP arrests translation of these proteins is relaxed in the presence of microsomal membranes. We also demonstrate that two previously described populations of membrane- associated ribosomes, distinguished by their sensitivity to high salt or EDTA extraction, correspond to ribosomes that have synthesized differing lengths of the nascent polypeptide. This analysis has revealed that nascent chain insertion into the membrane begins at distinct points for different presecretory proteins.     

Signal recognition particle causes a transient arrest in the biosynthesis of prepromelittin and mediates its translocation across mammalian endoplasmic reticulum

The translocation of prepromelittin (pPM) across mammalian endoplasmic reticulum was studied in both wheat germ and reticulocyte lysate. In the wheat germ system, signal recognition particle (SRP) caused a transient arrest in the synthesis of pPM. This was indicated by a slowdown in the rate of synthesis of pPM in the presence of SRP. The arrest was specific, dependent on the concentration of SRP, and more effective at early incubation time. In a tightly synchronized translation system, SRP had no apparent effect on the elongation of pPM, indicating that the effect of SRP on pPM chain synthesis might be at the final stages of chain elongation and release from the ribosome. This was reflected in a transient accumulation of pPM as peptidyl tRNA. Because pPM is composed of only 70 amino acids, arrest by SRP may be very close to chain termination. Arrest at this stage of chain synthesis seems to be unstable and the nascent chain gets terminated and released from the ribosome after a transient delay. The translocation of pPM was shown to be dependent on both SRP and docking protein. The difference in the translocation efficiency of pPM in reticulocyte and wheat germ lysates may reflect a difference in the targeting process in the two systems.     

Transient interaction of cpSRP54 with elongating nascent chains of the chloroplast-encoded D1 protein; 'cpSRP54 caught in the act'

The signal recognition particle (SRP) in bacteria and endoplasmic reticulum is involved in co-translational targeting. Plastids contain cpSRP54 and cpSRP43, unique to plants, but lack a SRP RNA molecule. A role for cpSRP in biogenesis of plastid-encoded membrane proteins has not been firmly established yet. In this study, a transient interaction between cpSRP54 and elongating D1 protein was observed using a homologous chloroplast translation system. Using the novel approach of cross-linking at different time points during elongation of full-length D1 protein, we showed that cpSRP54 interacts strongly with the elongating nascent chain forming two distinct cross-linked products. However, this interaction did not lead to an elongation arrest and cpSRP54 was released from the nascent chains, once they were longer than approximately 14 kDa. Detailed mutant analysis showed that the cpSRP54 interaction occurred via the first transmembrane domain, which could be replaced by other hydrophobic domains of more than 10 amino acids.     

The affinity of signal recognition particle for presecretory proteins is dependent on nascent chain length.

We have developed an assay in which incomplete preprolactin chains of varying lengths are targeted to the endoplasmic reticulum (ER) membrane in an elongation independent manner. The reaction had the same molecular requirements as nascent chain translocation across the ER membrane, namely, it was signal recognition particle (SRP) dependent, and required the nascent chain to be present as peptidyl tRNA (i.e. most likely ribosome associated) and to have its signal sequence exposed outside the ribosome. We found that the efficiency of the targeting reaction dropped dramatically as the chains grew longer than 140 amino acids in length, which probably reflected a decrease in affinity of the nascent chain-ribosome complex for SRP. Thus at physiological SRP concentrations (10 nM) there appears a sharp cut-off point in the ability of these chains to be targeted, while at high SRP concentrations (270 nM) all chains could be targeted. In kinetic experiments, high concentrations of SRP were found to change the time in elongation after which translocation of the nascent polypeptide could no longer occur.     

A truncation in the 14 kDa protein of the signal recognition particle leads to tertiary structure changes in the RNA and abolishes the elongation arrest activity of the particle.

The signal recognition particle (SRP) provides the molecular link between synthesis of polypeptides and their concomitant translocation into the endoplasmic reticulum. During targeting, SRP arrests or delays elongation of the nascent chain, thereby presumably ensuring a high translocation efficiency. Components of the Alu domain, SRP9/14 and the Alu sequences of SRP RNA, have been suggested to play a role in the elongation arrest function of SRP. We generated a truncated SRP14 protein, SRP14-20C, which forms, together with SRP9, a stable complex with SRP RNA. However, particles reconstituted with SRP9/14-20C, RC(9/14-20C), completely lack elongation arrest activity. RC(9/14-20C) particles have intact signal recognition, targeting and ribosome binding activities. SRP9/14-20C therefore only impairs interactions with the ribosome that are required to effect elongation arrest. This result provides evidence that direct interactions between the Alu domain components and the ribosome are required for this function. Furthermore, SRP9/14-20C binding to SRP RNA results in tertiary structure changes in the RNA. Our results strongly indicate that these changes account for the negative effect of SRP14 truncation on elongation arrest, thus revealing a critical role of the RNA in this function.     

Formation of a functional ribosome-membrane junction during translocation requires the participation of a GTP-binding protein

The requirement for ribonucleotides and ribonucleotide hydrolysis was examined at several distinct points during translocation of a secretory protein across the endoplasmic reticulum. We monitored binding of in vitro-assembled polysomes to microsomal membranes after removal of ATP and GTP. Ribonucleotides were not required for the initial low salt- insensitive attachment of the ribosome to the membrane. However, without ribonucleotides the nascent secretory chains were sensitive to protease digestion and were readily extracted from the membrane with either EDTA or 0.5 M KOAc. In contrast, nascent chains resisted extraction with either EDTA or 0.5 M KOAc and were insensitive to protease digestion after addition of GTP or nonhydrolyzable GTP analogues. Translocation of the nascent secretory polypeptide was detected only when ribosome binding was conducted in the presence of GTP. Thus, translocation-competent binding of the ribosome to the membrane requires the participation of a novel GTP-binding protein in addition to the signal recognition particle and the signal recognition particle receptor. The second event we examined was translocation and processing of a truncated secretory polypeptide. Membrane-bound polysomes bearing an 86-residue nascent chain were generated by translation of a truncated preprolactin mRNA. Ribonucleotide- independent translocation of the polypeptide was detected by cleavage of the 30-residue signal sequence after puromycin termination. Nascent chain transport, per se, is apparently dependent upon neither ribonucleotide hydrolysis nor continued elongation of the polypeptide once a functional ribosome-membrane junction has been established.     

SRP keeps polypeptides translocation-competent by slowing translation to match limiting ER-targeting sites

SRP is essential for targeting nascent chains to the endoplasmic reticulum, and it delays nascent chain elongation in cell-free translation systems. However, the significance of this function has remained unclear. We show that efficient protein translocation into the ER is incompatible with normal cellular translation rates due to rate-limiting concentrations of SRP receptor (SR). We complemented mammalian cells depleted of SRP14 by expressing mutant versions of the protein lacking the elongation arrest function. The absence of a delay caused inefficient targeting of preproteins leading to defects in secretion, depletion of proteins in the endogenous membranes, and reduced cell growth. The detrimental effects were reversed by either reducing the cellular protein synthesis rate or increasing SR expression. SRP therefore ensures that nascent chains remain translocation competent during the targeting time window dictated by SR. Since SRP-signal sequence affinities vary, the delay may also regulate which proteins are preferentially targeted.     

Protein translocation across the endoplasmic reticulum. II. Isolation and characterization of the signal recognition particle receptor

The signal recognition particle (SRP)-mediated elongation arrest of the synthesis of nascent secretory proteins can be released by salt- extracted rough microsomal membranes (Walter, P., and G. Blobel, 1981, J. Cell Biol, 91:557-561). Both the arrest-releasing activity and the signal peptidase activity were solubilized from rough microsomal membranes using the nonionic detergent Nikkol in conjunction with 250 mM KOAc. Chromatography of this extract on SRP-Sepharose separated the arrest-releasing activity from the signal peptidase activity. Further purification of the arrest-releasing activity using sucrose gradient centrifugation allowed the identification of a 72,000-dalton polypeptide as the protein responsible for the activity. Based upon its affinity for SRP, we refer to the 72,000-dalton protein as the SRP receptor. A 60,000-dalton protein fragment (Meyer, D. I., and B. Dobberstein, 1980, J. Cell Biol., 87:503-508) that had been shown previously to reconstitute the translocation activity of protease- digested membranes, was shown here by peptide mapping and immunological criteria to be derived from the SRP receptor. Findings that are in part similar, and in part different from these reported here and in our preceding paper were made independently (Meyer, D. I., E. Krause, and B. Dobberstein, 1982, Nature (Lond.). 297:647-650) and the term "docking protein" was proposed for the SRP receptor. A lower membrane content of both SRP and the SRP receptor than that of membrane bound ribosomes suggests that the SRP-SRP receptor interaction may exist transiently during the formation of a ribosome-membrane junction and during translocation.     

Co-translational protein targeting catalyzed by the Escherichia coli signal recognition particle and its receptor.

The Ffh-4.5S ribonucleoprotein particle (RNP) and FtsY from Escherichia coli are homologous to essential components of the mammalian signal recognition particle (SRP) and SRP receptor, respectively. The ability of these E. coli components to function in a bona fide co-translational targeting pathway remains unclear. Here we demonstrate that the Ffh-4.5S RNP and FtsY can efficiently replace their mammalian counterparts in targeting nascent secretory proteins to microsomal membranes in vitro. Targeting in the heterologous system requires a hydrophobic signal sequence, utilizes GTP and, moreover, occurs co-translationally. Unlike mammalian SRP, however, the Ffh-4.5S RNP is unable to arrest translational elongation, which results in a narrow time window for the ribosome nascent chain to interact productively with the membrane-bound translocation machinery. The highly negatively charged N-terminal domain of FtsY, which is a conserved feature among prokaryotic SRP receptor homologs, is important for translocation and acts to localize the protein to the membrane. Our data illustrate the extreme functional conservation between prokaryotic and eukaryotic SRP and SRP receptors and suggest that the basic mechanism of co-translational protein targeting is conserved between bacteria and mammals.     

Signal recognition particle mediates a transient elongation arrest of preprolactin in reticulocyte lysate

Signal recognition particle (SRP) is a ribonucleoprotein that functions in the targeting of ribosomes synthesizing presecretory proteins to the ER. SRP binds to the signal sequence as it emerges from the ribosome, and in wheat germ extracts, arrests further elongation. The translation arrest is released when SRP interacts with its receptor on the ER membrane. We show that the delay of elongation mediated by SRP is not unique to wheat germ translation extracts. Addition of mammalian SRP to reticulocyte lysates resulted in a delay of preprolactin synthesis due to increased ribosome pausing at specific sites on preprolactin mRNA. Addition of canine pancreatic microsomal membranes to reticulocyte lysates resulted in an acceleration of preprolactin synthesis, suggesting that the endogenous SRP present in the reticulocyte lysate also delays synthesis of secretory proteins.     

Targeting and insertion of heterologous membrane proteins in E. coli

Membrane targeting and insertion of the archaeal Halobacter halobium proton pump bacterioopsin (Bop) and the human melanocortin 4 receptor (MC(4)R) were studied in vitro, using E. coli components for protein synthesis, membrane targeting and insertion. These heterologous proteins are targeted to E. coli membranes in a signal recognition particle (SRP) dependent manner and inserted into the membrane co-translationally. Furthermore, we demonstrate that nascent chains of Bop and MC(4)R first interact with SecY and then with YidC as they move through the translocon. Our results suggest that the initial stages of membrane targeting and insertion of heterologous proteins in E. coli proceed by the pathway used for native E. coli membrane proteins. No significant pausing of protein elongation was observed in the presence of E. coli SRP, in line with the suggestion that translational arrest requires an Alu domain, which is absent in SRP from E. coli.     

The heterodimeric subunit SRP9/14 of the signal recognition particle functions as permuted single polypeptide chain.

The targeting of nascent polypeptide chains to the endoplasmic reticulum is mediated by a cytoplasmic ribonucleoprotein, the signal recognition particle (SRP). The 9 kD (SRP9) and the 14 kD (SRP14) subunits of SRP are required to confer elongation arrest activity to the particle. SRP9 and SRP14 form a heterodimer which specifically binds to SRP RNA. We have constructed cDNAs that encode single polypeptide chains comprising SRP9 and SRP14 sequences in the two possible permutations linked by a 17 amino acid peptide. We found that both fusion proteins specifically bound to SRP RNA as monomeric molecules folded into a heterodimer-like structure. Our results corroborate the previous hypothesis that the authentic heterodimer binds to SRP RNA in equimolar ratio. In addition, both fusion proteins conferred elongation arrest activity to SRP(-9/14), which lacks this function, and one fusion protein could functionally replace the heterodimer in the translocation assay. Thus, the normal N-and C-termini of both proteins have no essential role in folding, RNA-binding and in mediating the biological activities. The possibility to express the heterodimeric complex as a single polypeptide chain facilitates the analysis of its functions and its structure in vivo and in vitro.