Signal Recognition Particle and SecA Cooperate during Export of Secretory Proteins with Highly Hydrophobic Signal Sequences

The Sec translocon of bacterial plasma membranes mediates the linear translocation of secretory proteins as well as the lateral integration of membrane proteins. Integration of many membrane proteins occurs co-translationally via the signal recognition particle (SRP)-dependent targeting of ribosome-associated nascent chains to the Sec translocon. In contrast, translocation of classical secretory proteins across the Sec translocon is a post-translational event requiring no SRP but the motor protein SecA. Secretory proteins were, however, reported to utilize SRP in addition to SecA, if the hydrophobicity of their signal sequences exceeds a certain threshold value. Here we have analyzed transport of this subgroup of secretory proteins across the Sec translocon employing an entirely defined in vitro system. We thus found SecA to be both necessary and sufficient for translocation of secretory proteins with hydrophobic signal sequences, whereas SRP and its receptor improved translocation efficiency. This SRP-mediated boost of translocation is likely due to the early capture of the hydrophobic signal sequence by SRP as revealed by site-specific photo cross-linking of ribosome nascent chain complexes.     

Signal sequence-dependent function of the TRAM protein during early phases of protein transport across the endoplasmic reticulum membrane

Cotranslational translocation of proteins across the mammalian ER membrane involves, in addition to the signal recognition particle receptor and the Sec61p complex, the translocating chain-associating membrane (TRAM) protein, the function of which is still poorly understood. Using reconstituted proteoliposomes, we show here that the translocation of most, but not all, secretory proteins requires the function of TRAM. Experiments with hybrid proteins demonstrate that the structure of the signal sequence determines whether or not TRAM is needed. Features that distinguish TRAM-dependent and -independent signal sequences include the length of their charged, NH2-terminal region and the structure of their hydrophobic core. In cases where TRAM is required for translocation, it is not needed for the initial interaction of the ribosome/nascent chain complex with the ER membrane but for a subsequent step inside the membrane in which the nascent chain is inserted into the translocation site in a protease-resistant manner. Thus, TRAM functions in a signal sequence-dependent manner at a critical, early phase of the translocation process.     

Regulation of ribosome detachment from the mammalian endoplasmic reticulum membrane

In current models, protein translocation in the endoplasmic reticulum (ER) occurs in the context of two cycles, the signal recognition particle (SRP) cycle and the ribosome cycle. Both SRP and ribosomes bind to the ER membrane as a consequence of the targeting process of translocation. Whereas SRP release from the ER membrane is regulated by the GTPase activities of SRP and the SRP receptor, ribosome release from the ER membrane is thought to occur in response to the termination of protein synthesis. We report that ER-bound ribosomes remain membrane-bound following the termination of protein synthesis and in the bound state can initiate the translation of secretory and cytoplasmic proteins. Two principal observations are reported. 1) Membrane-bound ribosomes engaged in the synthesis of proteins lacking a signal sequence are released from the ER membrane as ribosome-nascent polypeptide complexes. 2) Membrane-bound ribosomes translating secretory proteins can access the translocon in an SRP receptor-independent manner. We propose that ribosome release from the ER membrane occurs in the context of protein translation, with release occurring by default in the absence of productive nascent polypeptide-membrane interactions.     

Nascent secretory polypeptides synthesized on Escherichia coli ribosomes are not translocated across mammalian endoplasmic reticulum.

Cell-free protein-synthesizing systems from Escherichia coli and wheat germ were compared for their capacity to support the translocation of secretory proteins across microsomal membranes derived from mammalian endoplasmic reticulum. Three different secretory proteins, two of bacterial and one of eucaryotic origin, were tested in this respect. In all three cases a contrast between the results in the eucaryotic and procaryotic protein-synthesizing systems was revealed. Whereas the eucaryotic system, as expected, supported the translocation of nascent secretory proteins across the microsomal membranes, the procaryotic system failed to do so. This failure was not due to the absence of a translocation-promoting activity or the presence of a translocation-blocking activity in the procaryotic system. These results demonstrate a specificity in the requirement of components of the protein-synthesizing machinery for protein translocation. These components might participate in forming a functional ribosome-membrane junction during protein translocation. The nascent secretory chain alone is not sufficient for making this junction, which might involve the postulated binding of the ribosome to the signal recognition particle or another component of the membrane.     

Translocation of proteins across the endoplasmic reticulum. II. Signal recognition protein (SRP) mediates the selective binding to microsomal membranes of in-vitro-assembled polysomes synthesizing secretory protein

Translocation-competent microsomal membrane vesicles of dog pancreas were shown to selectively bind nascent, in vitro assembled polysomes synthesizing secretory protein (bovine prolactin) but not those synthesizing cytoplasmic protein (alpha and beta chain of rabbit globin). This selective polysome binding capacity was abolished when the microsomal vesicles were salt-extracted but was restored by an 11S protein (SRP, Signal Recognition Protein) previously purified from the salt-extract of microsomal vesicles (Walter and Blobel, 1980. Proc. Natl. Acad. Sci. U. S. A. 77:7112-7116). SRP-dependent polysome recognition and binding to the microsomal membrane was shown to be a prerequisite for chain translocation. Modification of SRP by N-ethyl maleimide abolished its ability to mediate nascent polysome binding to the microsomal vesicles. Likewise, polysome binding to the microsomal membrane was largely abolished when beta-hydroxy leucine, a Leu analogue, was incorporated into nascent secretory polypeptides. The data in this and the preceding paper provide conclusive experimental evidence that chain translocation across the endoplasmic reticulum membrane is a receptor-mediated event and thus rule out proposals that chain translocation occurs spontaneously and without the mediation by proteins. Moreover, our data here demonstrate conclusively that the initial events that lead to translocation and provide for its specificity are protein-protein (signal sequence plus ribosome with SRP) and not protein-lipid (signal sequence with lipid bilayer) interactions.     

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.     

SecA interacts with secretory proteins by recognizing the positive charge at the amino terminus of the signal peptide in Escherichia coli

SecA is an acidic, peripheral membrane protein involved in the translocation of secretory proteins across the cytoplasmic membrane. The direct interaction of SecA with secretory proteins was demonstrated by means of chemical cross-linking with 1-ethyl-3-(3-dimethylaminoprophyl)carbodiimide. OmpF-Lpp, a model secretory protein, carries either an uncleavable or cleavable signal peptide, and mutant secretory proteins derived from uncleavable OmpF-Lpp were used as translocation substrates. The interaction was SecA-specific. None of the control proteins, which are as acidic as SecA, was cross-linked with uncleavable OmpF-Lpp. The interaction was signal peptide-dependent. The interaction was increasingly enhanced as the number of positively charged amino acid residues at the amino-terminal region of the signal peptide was increased, irrespective of the species of amino acid residues donating the charge. Finally, parallelism was observed between the efficiency of interaction and that of translocation among mutant secretory proteins. It is suggested that precursors of secretory proteins interact with SecA to initiate the translocation reaction.     

Protein translocation across wheat germ microsomal membranes requires an SRP-like component

Different wheat germ extracts were tested for the presence of membranes capable of translocating and processing nascent secretory proteins. One lysate was found in which nascent prehuman-placental lactogen (phPL) was translocated and processed to mature human placental lactogen (hPL). Processing was found to occur concomitant with translocation across membranes. Translocation across the wheat germ membrane required a component which is similar to the mammalian signal recognition particle (SRP). It bound to DEAE–Sepharose, had a sedimentation coefficient of 11S and contained a 7S RNA. In addition to hPL, the plant protein zein and the bacterial protein β-lactamase were translocated across and processed by wheat germ membranes. Transport was found to occur only co-translationally. Our results show that the wheat germ protein translocation system is similar to the mammalian one. Unlike the mammalian SRP, the particle purified from wheat germ did not arrest elongation of nascent secretory proteins.     

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.     

Systematic probing of the environment of a translocating secretory protein during translocation through the ER membrane.

We have extended a previously developed photo-crosslinking approach to systematically probe the protein environment of the secretory protein preprolactin, trapped during its transfer through the endoplasmic reticulum membrane. Single photoreactive groups were placed at various positions of nascent polypeptide chains of various length, corresponding to different stages of the transport process, and photo-crosslinks to membrane proteins were analyzed. In all cases, the polypeptide segment extending from the ribosome was found to be located in a membrane environment that is formed almost exclusively from Sec61 alpha, the multi-spanning subunit of the Sec61p complex that is essential for translocation. At early stages of the translocation process, before cleavage of the signal sequence, almost the entire nascent chain emerged from the ribosome contacts Sec61 alpha. The 'translocating chain-associating membrane' protein interacts mainly with the region of the signal sequence preceding its hydrophobic core. Our results suggest that the nascent chain is transferred directly from the ribosome into a protein-conducting channel, the major constituent of which is Sec61 alpha.     

SRP-dependent co-translational targeting and SecA-dependent translocation analyzed as individual steps in the export of a bacterial protein

Recently it has been recognized that the signal recognition particle (SRP) of Escherichia coli represents a specific targeting device for hydrophobic inner membrane proteins. It has remained unclear, however, whether the bacterial SRP functions in concert with SecA, which is required for the translocation of secretory proteins across the inner membrane. Here, we have analyzed a hybrid protein constructed by fusing the signal anchor sequence of an SRP-dependent inner membrane protein (MtlA) to the mature part of an exclusively SecA-requiring secretory protein (OmpA). We show that the signal anchor sequence of MtlA confers the novel properties onto nascent chains of OmpA of being co-translationally recognized and targeted to SecY by SRP. Once targeted to SecY, ribosome-associated nascent chains of the hybrid protein, however, remain untranslocated unless SecA is present. These results indicate that SRP and SecA cooperate in a sequential, non-overlapping manner in the topogenesis of those membrane proteins which, in addition to a signal anchor sequence, harbor a substantial hydrophilic domain to be translocated into the periplasm.     

Identification of signal sequence binding proteins integrated into the rough endoplasmic reticulum membrane.

An azidophenacyl derivative of a chemically synthesized consensus signal peptide has been prepared. The peptide, when photoactivated in the presence of rough or high-salt-stripped microsomes from pancreas, leads to inhibition of their activity in cotranslational processing of secretory pre-proteins translated from their mRNA in vitro. The peptide binds specifically with high affinity to components in the microsomal membranes from pancreas and liver, and photoreaction of a radioactive form of the azidophenacyl derivative leads to covalent linkage to yield two closely related radiolabelled proteins of Mr about 45,000. These proteins are integrated into the membrane, with large 30,000-Mr domains embedded into the phospholipid bilayer to which the signal peptide binds. A smaller, endopeptidase-sensitive, domain is exposed on the cytoplasmic surface of the microsomal vesicles. The specificity and selectivity of the binding of azidophenacyl-derivatized consensus signal peptide was demonstrated by concentration-dependent inhibition of photolabelling by the 'cold' synthetic consensus signal peptide and by a natural internal signal sequence cleaved and isolated from ovalbumin. The properties of the labelled 45,000-Mr protein-signal peptide complexes, i.e. mass, pI, ease of dissociation from the membrane by detergent or salts and immunological properties, distinguish them from other proteins, e.g. subunits of signal recognition particle, docking protein and signal peptidase, already known to be involved in targetting and processing of nascent secretory proteins at the rough endoplasmic reticulum membrane. Although the 45,000-Mr signal peptide binding protein displays properties similar to those of the signal peptidase, a component of the endoplasmic reticulum, the azido-derivatized consensus signal peptide does not interact with it. It is proposed that the endoplasmic reticulum proteins with which the azidophenacyl-derivatized consensus signal peptide interacts to yield the 45,000-Mr adducts may act as receptors for signals in nascent secretory pre-proteins in transduction of changes in the endoplasmic reticulum which bring about translocation of secretory protein across the membrane.     

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.     

Regulation of the Ribosome–Membrane Junction at Early Stages of Presecretory Protein Translocation in the Mammalian Endoplasmic Reticulum

A series of fusion protein constructs were designed to investigate the contribution of secretory nascent chains to regulation of the ribosome–membrane junction in the mammalian endoplasmic reticulum. As a component of these studies, the membrane topology of the signal sequence was determined at stages of protein translocation immediately after targeting and before signal sequence cleavage. Truncated translation products were used to delimit the analysis to defined stages of translocation.

In a study of secretory protein precursors, formation of a protease-resistant ribosome–membrane junction, currently thought to define the pathway of the translocating nascent chain, was observed to be precursor- and stage-dependent. Analysis of the binding of early intermediates indicated that the nascent chain was bound to the membrane independent of the ribosome, and that the binding was predominately electrostatic. The membrane topology of the signal sequence was determined as a function of the stage of translocation, and was found to be identical for all assayed intermediates. Unexpectedly, the hydrophobic core of the signal sequence was observed to be accessible to the cytosolic face of the membrane at stages of translocation immediately after targeting as well as stages before signal sequence cleavage. Removal of the ribosome from bound intermediates did not disrupt subsequent translocation, suggesting that the active state of the protein-conducting channel is maintained in the absence of the bound ribosome. A model describing a potential mode of regulation of the ribosome–membrane junction by the nascent chain is presented.

     

Evidence for a two-step mechanism involved in assembly of functional signal recognition particle receptor

The signal recognition particle (SRP) and SRP receptor act sequentially to target nascent secretory proteins to the membrane of the ER. The SRP receptor consists of two subunits, SR alpha and SR beta, both tightly associated with the ER membrane. To examine the biogenesis of the SRP receptor we have developed a cell-free assay system that reconstitutes SR alpha membrane assembly and permits both anchoring and functional properties to be assayed independently. Our experiments reveal a mechanism involving at least two distinct steps, targeting to the ER and anchoring of the targeted molecule on the cytoplasmic face of the membrane. Both steps can be reconstituted in vitro to restore translocation activity to ER microsomes inactivated by alkylation with N-ethyl-maleimide. The characteristics elucidated for this pathway distinguish it from SRP-dependent targeting of secretory proteins, SRP-independent ER translocation of proteins such as prepromellitin, and direct insertion mechanisms of the type exemplified by cytochrome b5.     

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.     

Protein translocation across the endoplasmic reticulum membrane: identification by photocross-linking of a 39-kD integral membrane glycoprotein as part of a putative translocation tunnel

The molecular environment of secretory proteins during translocation across the ER membrane was examined by photocross-linking. Nascent preprolactin chains of various lengths, synthesized by in vitro translation of truncated messenger RNAs in the presence of N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA, signal recognition particle, and microsomal membranes, were used to position photoreactive probes at various locations within the membrane. Upon photolysis, each nascent chain species was cross-linked to an integral membrane glycoprotein with a deduced mass of 39 kD (mp39) via photoreactive lysines located in either the signal sequence or the mature prolactin sequence. Thus, different portions of the nascent preprolactin chain are in close proximity to the same membrane protein during the course of translocation, and mp39 therefore appears to be part of the translocon, the specific site of protein translocation across the ER membrane. The similarity of the molecular and cross-linking properties of mp39 and the glyco-protein previously identified as a signal sequence receptor (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature [Lond.]. 328: 830-833) suggests that these two proteins may be identical. Our data indicate, however, that mp39 does not (or not only) function as a signal sequence receptor, but rather may be part of a putative translocation tunnel.     

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.     

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.     

Amino acid sequences of transport peptides associated with canine exocrine pancreatic proteins

Using microsequencing techniques and proteins labeled in vitro with tritiated amino acids we have obtained the following NH2-terminal sequences for six canine pancreatic presecretory proteins: pretrypsinogen 1, pretrypsinogen 2+3, prechymotrypsinogen 2, preproelastase1, preporcarboxypeptidase A1, preamylase. Points of cleavage by the transport peptidase, indicated by the vertical arrows, were located from sequences of authentic products synthesized in the presence of membranes of the rough endoplasmic reticulum. All of the identified residues in the pancreatic transport peptides are hydrophobic. Predictions of secondary structure were calculated for each of the transport peptides. The data indicated neither a common primary of secondary structure which could be interpreted as the signal for functional binding of the nascent presecretory protein to the rough endoplasmic reticulum membrane. These findings suggest that the initial interaction with the membrane or membrane receptor may depend in part, on the hydrophobic nature of the transport peptides. Five of the presecretory proteins showed a region with a high probability of forming a beta-turn immediately following the cleavage point. This feature may give the nascent peptide a region of flexibility that would facilitate both its insertion as a loop structure into the membrane and its cleavage by the transport peptidase. The sequences of authentic secretory products derived from a variety of pancreatic tissues suggest that hydrophilic residues are required immediately following the cleavage point in order to allow translocation of the nascent polypeptide chains across the membrane.