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.     

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 secretory proteins across the microsomal membrane occurs through an environment accessible to aqueous perturbants

We have characterized the association of a nascent secretory protein with the microsomal membrane at two distinct stages in cell-free synthesis and translocation. Stage one corresponded to a nascent chain of approximately 70 residues generated via elongation arrest by the signal recognition particle (SRP). Binding to microsomal membranes occurred independently of chain elongation and required SRP receptor. Following binding, the 70-mer remained attached to the membrane after extraction of the ribosome. However, protein denaturants (4 M urea or alkaline pH) extracted the 70-mer from the membrane. Stage two of synthesis corresponded to nascent chains of approximately 158 residues generated by oligonucleotide-mediated hybrid arrest of translation. Again, these partially translocated nascent chains were extracted by 4 M urea. Therefore, the initial interaction of the signal sequence with the membrane as well as subsequent chain conductance occur in a microenvironment that is accessible to aqueous reagents. Thus, both processes probably require integral membrane proteins.     

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 Alu domain occupies a defined site at the ribosomal subunit interface upon signal sequence recognition

The eukaryotic signal recognition particle (SRP) is essential for cotranslational targeting of proteins to the endoplasmic reticulum (ER). The SRP Alu domain is specifically required for delaying nascent chain elongation upon signal sequence recognition by SRP and was therefore proposed to interact directly with ribosomes. Using protein cross-linking, we provide experimental evidence that the Alu binding protein SRP14 is in close physical proximity of several ribosomal proteins in functional complexes. Cross-linking occurs even in the absence of a signal sequence in the nascent chain demonstrating that SRP can bind to all translating ribosomes and that close contacts between the Alu domain and the ribosome are independent of elongation arrest activity. Without a signal sequence, SRP14 cross-links predominantly to a protein of the large subunit. Upon signal sequence recognition, certain cross-linked products become detectable or more abundant revealing a change in the Alu domain-ribosome interface. At this stage, the Alu domain of SRP is located at the ribosomal subunit interface since SRP14 can be cross-linked to proteins from the large and small ribosomal subunits. Hence, these studies reveal differential modes of SRP-ribosome interactions mediated by the Alu domain.     

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.     

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.     

Elongation arrest is a physiologically important function of signal recognition particle

Signal recognition particle (SRP) targets proteins for co-translational insertion through or into the endoplasmic reticulum membrane. Mammalian SRP slows nascent chain elongation by the ribosome during targeting in vitro. This 'elongation arrest' activity requires the SRP9/14 subunit of the particle and interactions of the C-terminus of SRP14. We have purified SRP from Saccharomyces cerevisiae and demonstrated that it too has elongation arrest activity. A yeast SRP containing Srp14p truncated at its C-terminus (delta C29) did not maintain elongation arrest, was substantially deficient in promoting translocation and interfered with targeting by wild-type SRP. In vivo, this mutation conferred a constitutive defect in the coupling of protein translation and translocation and temperature-sensitive growth, but only a slight defect in protein translocation. In combination, these data indicate that the primary defect in SRP delta C29 is in elongation arrest, and that this is a physiologically important and conserved function of eukaryotic SRP.     

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.     

The signal recognition particle binds to protein L23 at the peptide exit of the Escherichia coli ribosome

The signal recognition particle (SRP) from Escherichia coli, composed of Ffh protein and 4.5S RNA, mediates membrane targeting of translating ribosomes displaying a signal or signal-anchor sequence. SRP binds at the peptide exit of the large ribosomal subunit. Structural details of the interaction are not known. Here, the position of Ffh or SRP on the ribosome was probed by using site-specific UV-induced crosslinking by p-azidophenacyl bromide (AzP) attached to a number of cysteine residues engineered into surface positions of Ffh. Efficient crosslinking to vacant ribosomes took place from two positions (AzP17 and AzP25) in the N domain of Ffh, both with Ffh and SRP. Both AzP17 and AzP25 were predominantly crosslinked to ribosomal protein L23 that is located at the peptide exit of the 50S subunit. The SRP receptor, FtsY, did not change the crosslink pattern, whereas the presence of a nascent signal peptide on the ribosome resulted in a second crosslink between Ffh(AzP17) and protein L23, indicating that binding to the nascent signal peptide induced a slightly different arrangement of SRP on the ribosome. These results indicate a model of the topographical arrangement of SRP at the peptide exit of the 50S ribosomal subunit.     

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.     

Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in-vitro-assembled polysomes synthesizing secretory protein

An 11S protein composed of six polypeptide chains was previously purified from a salt extract of dog pancreas microsomal membranes and shown to be required for translocation of nascent secretory protein across the microsomal membrane (Wistar and Blobel 1980 Proc. Natl. Acad. Sci. U. S. A. 77:7112-7116). This 11S protein, termed signal recognition protein (SRP), has been shown here (a) to inhibit translation in the wheat germ cell-free system selectively of mRNA for secretory protein (bovine preprolactin) but not of mRNA for cytoplasmic protein (alpha and beta chain of rabbit globin); (b) to bind with relatively low affinity (apparent KD less than 5 x 10(-5)) to monomeric wheat germ ribosomes; and (c) to bind selectively and with 6,000-fold higher affinity (apparent KD less than 8 x 10(-9)) to wheat germ ribosomes engaged in the synthesis of secretory protein but not to those engaged in the synthesis of cytoplasmic protein. Low- and high- affinity binding as well as the selective translation-inhibitory effect were abolished after modification of SRP by N-ethyl maleimide. High- affinity binding and the selective translation-inhibitory effect of SRP were largely abolished when the leucine (Leu) analogue beta-hydroxy leucine was incorporated into the nascent secretory polypeptide.     

GTPase domain of the 54-kD subunit of the mammalian signal recognition particle is required for protein translocation but not for signal sequence binding

The 54-kD subunit of the signal recognition particle (SRP54) binds to signal sequences of nascent secretory and transmembrane proteins. SRP54 consists of two separable domains, a 33-kD amino-terminal domain that contains a GTP-binding site (SRP54G) and a 22-kD carboxy-terminal domain (SRP54M) containing binding sites for both the signal sequence and SRP RNA. To examine the function of the two domains in more detail, we have purified SRP54M and used it to assemble a partial SRP that lacks the amino-terminal domain of SRP54 [SRP(-54G)]. This particle recognized signal sequences in two independent assays, albeit less efficiently than intact SRP. Analysis of the signal sequence binding activity of free SRP54 and SRP54M supports the conclusion that SRP54M binds signal sequences with lower affinity than the intact protein. In contrast, when SRP(-54G) was assayed for its ability to promote the translocation of preprolactin across microsomal membranes, it was completely inactive, apparently because it was unable to interact normally with the SRP receptor. These results imply that SRP54G plays an essential role in SRP-mediated targeting of nascent chain-ribosome complexes to the ER membrane and also influences signal sequence recognition, possibly by promoting a tighter association between signal sequences and SRP54M.     

The methionine-rich domain of the 54 kDa subunit of signal recognition particle is sufficient for the interaction with signal sequences.

The signal recognition particle (SRP) binds to signal sequences when they emerge from a translating ribosome and targets the complex of ribosome, nascent chain and SRP to the membrane of the rough endoplasmic reticulum (rER) allowing the co-translational translocation of the nascent chain. By photo-crosslinking it has been shown that the signal sequence of preprolactin (PPL) only interacts with the methionine-rich (M) domain of the 54 kDa protein subunit (SRP54) of SRP. Here we show that (i) a signal-anchor sequence is likewise crosslinked only to the methionine-rich domain of SRP54, (ii) free SRP54 can interact with signal sequences independently of the other components of SRP, (iii) its M domain suffices to perform this function, and (iv) an essentially intact M domain is required for signal sequence recognition. Alkylation of the N+G domain in intact SRP54 with N-ethyl maleimide (NEM), but not after cleavage with V8 protease, prevents the binding of a signal sequence to the M domain. This suggests a proximity between the N+G and M domains of SRP54 and raises the possibility that the role of the N+G domain may be to regulate the binding and/or the release of signal sequences.     

Signal Recognition Particle-ribosome Binding Is Sensitive to Nascent Chain Length

The signal recognition particle (SRP) directs ribosome-nascent chain complexes (RNCs) displaying signal sequences to protein translocation channels in the plasma membrane of prokaryotes and endoplasmic reticulum of eukaryotes. It was initially proposed that SRP binds the signal sequence when it emerges from an RNC and that successful binding becomes impaired as translation extends the nascent chain, moving the signal sequence away from SRP on the ribosomal surface. Later studies drew this simple model into question, proposing that SRP binding is unaffected by nascent chain length. Here, we reinvestigate this issue using two novel and independent fluorescence resonance energy transfer assays. We show that the arrival and dissociation rates of SRP binding to RNCs vary according to nascent chain length, resulting in the highest affinity shortly after a functional signal sequence emerges from the ribosome. Moreover, we show that SRP binds RNCs in multiple and interconverting conformations, and that conversely, RNCs exist in two conformations distinguished by SRP interaction kinetics.     

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.     

Elongation arrest by SecM via a cascade of ribosomal RNA rearrangements

In E. coli, the SecM nascent polypeptide causes elongation arrest, while interacting with 23S RNA bases A2058 and A749-753 in the exit tunnel of the large ribosomal subunit. We compared atomic models fitted by real-space refinement into cryo-electron microscopy reconstructions of a pretranslocational and SecM-stalled E. coli ribosome complex. A cascade of RNA rearrangements propagates from the exit tunnel throughout the large subunit, affecting intersubunit bridges and tRNA positions, which in turn reorient small subunit RNA elements. Elongation arrest could result from the inhibition of mRNA.(tRNAs) translocation, E site tRNA egress, and perhaps translation factor activation at the GTPase-associated center. Our study suggests that the specific secondary and tertiary arrangement of ribosomal RNA provides the basis for internal signal transduction within the ribosome. Thus, the ribosome may itself have the ability to regulate its progression through translation by modulating its structure and consequently its receptivity to activation by cofactors.     

Mathematical modeling of the effects of the signal recognition particle on translation and translocation of proteins across the endoplasmic reticulum membrane

The kinetics of the signal recognition particle(SRP)-mediated process of protein translocation across the endoplasmic reticulum membrane was studied by mathematical modeling and complementary experiments. The following results were obtained. (1) A model according to which SRP directs the ribosome, rather than the mRNA, to the membrane is supported by experiments designed to discriminate between the two alternatives. (2) This model describes both steady-state and synchronized translation experiments and makes a number of predictions. (3) The interaction between a nascent protein and SRP may be described by two parameters: (i) a binding constant which can be attributed to the structure of the signal peptide, and (ii) the size of the "SRP-window", i.e. the distance between the first and the last site on the polypeptide chain that can interact with SRP. For preprolactin a binding constant of 1 to 2.5 nmol-1l was estimated. Modeling of the synchronized synthesis of ovalbumin indicates that it has a much weaker binding constant than preprolactin (approximately 0.25 nmol-1l) although we cannot exclude the possibility that the SRP-window may be also smaller. (4) A better understanding of the molecular effects of SRP on translation and translocation through the rough endoplasmic reticulum membrane has been achieved. Inhibition of the steady-state rate of translation by SRP requires a stoichiometric interaction of SRP with ribosomes carrying nascent polypeptide chains and will occur only when ribosomes are piled up back to the initiation site. Translocation, on the other hand, requires only the catalytic action of SRP and is determined by the local concentration of protein-synthesizing ribosomes accumulated at the site(s) of SRP interaction. As a consequence, translational inhibition by SRP may sometimes fail to occur, depending either on the type of protein or on experimental conditions, such as a high mRNA concentration, even if translocation can be demonstrated. (5) A rough extrapolation to the conditions in vivo indicates that all synthesized polypeptide chains destined for translocation across or integration into the endoplasmic reticulum membrane are indeed quantitatively translocated and that no translational inhibition occurs.     

Photoaffinity labeling of dog pancreas microsomes with 8-azido-ATP inhibits association of nascent preprolactin with the signal sequence receptor complex.

Transport of bovine preprolactin into dog pancreas microsomes involves a microsomal protein which is sensitive to photoaffinity labeling with azido-ATP and which is distinct from the ATP-binding protein, immunoglobulin heavy chain binding protein. Here we addressed the question of what stage of preprolactin transport is affected. Thus a nascent presecretory protein which is related to preprolactin, termed ppl-86mer, was employed. Here we show that the nascent preprolactin did not become associated with the alpha-subunit of the signal sequence receptor complex after photoaffinity labeling of microsomes with azido-ATP. Therefore, we conclude that the microsomal protein which is sensitive to photoaffinity labeling with azido-ATP acts prior to the signal sequence receptor complex.