The signal sequence receptor has a second subunit and is part of a translocation complex in the endoplasmic reticulum as probed by bifunctional reagents

Bifunctional cross-linking reagents were used to probe the protein environment in the ER membrane of the signal sequence receptor (SSR), a 24-kD integral membrane glycoprotein (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature [Lond.]. 328:830-833). The proximity of several polypeptides was demonstrated. A 22-kD glycoprotein was identified tightly bound to the 34-kD SSR even after membrane solubilization. The 34-kD polypeptide, now termed alpha SSR, and the 22-kD polypeptide, the beta SSR, represent a heterodimer. We report on the sequence of the beta SSR, its membrane topology, and on the mechanism of its integration into the membrane. Cross-linking also produced dimers of the alpha-subunit of the SSR indicating that oligomers of the SSR exist in the ER membrane. Various bifunctional cross-linking reagents were used to study the relation to ER membrane proteins of nascent chains of preprolactin and beta-lactamase at different stages of their translocation through the membrane. The predominant cross-linked products obtained in high yields contained the alpha SSR, indicating in conjunction with previous results that it is a major membrane protein in the neighborhood of translocating nascent chains of secretory proteins. The results support the existence of a translocon, a translocation complex involving the SSR, which constitutes the specific site of protein translocation across the ER 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.     

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.     

Nascent membrane and presecretory proteins synthesized in Escherichia coli associate with signal recognition particle and trigger factor

The Escherichia coli signal recognition particle (SRP) and trigger factor are cytoplasmic factors that interact with short nascent polypeptides of presecretory and membrane proteins produced in a heterologous in vitro translation system. In this study, we use an E. coli in vitro translation system in combination with bifunctional cross-linking reagents to investigate these interactions in more detail in a homologous environment. Using this approach, the direct interaction of SRP with nascent polypeptides that expose particularly hydrophobic targeting signals is demonstrated, suggesting that inner membrane proteins are the primary physiological substrate of the E. coli SRP. Evidence is presented that the overproduction of proteins that expose hydrophobic polypeptide stretches, titrates SRP. In addition, trigger factor is efficiently cross-linked to nascent polypeptides of different length and nature, some as short as 57 amino acid residues, indicating that it is positioned near the nascent chain exit site on the E. coli ribosome.     

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.     

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 beta subunit of the signal recognition particle receptor is a transmembrane GTPase that anchors the alpha subunit, a peripheral membrane GTPase, to the endoplasmic reticulum membrane

The signal recognition particle receptor (SR) is required for the cotranslational targeting of both secretory and membrane proteins to the endoplasmic reticulum (ER) membrane. During targeting, the SR interacts with the signal recognition particle (SRP) which is bound to the signal sequence of the nascent protein chain. This interaction catalyzes the GTP-dependent transfer of the nascent chain from SRP to the protein translocation apparatus in the ER membrane. The SR is a heterodimeric protein comprised of a 69-kD subunit (SR alpha) and a 30- kD subunit (SR beta) which are associated with the ER membrane in an unknown manner. SR alpha and the 54-kD subunits of SRP (SRP54) each contain related GTPase domains which are required for SR and SRP function. Molecular cloning and sequencing of a cDNA encoding SR beta revealed that SR beta is a transmembrane protein and, like SR alpha and SRP54, is a member of the GTPase superfamily. Although SR beta defines its own GTPase subfamily, it is distantly related to ARF and Sar1. Using UV cross-linking, we confirm that SR beta binds GTP specifically. Proteolytic digestion experiments show that SR alpha is required for the interaction of SRP with SR. SR alpha appears to be peripherally associated with the ER membrane, and we suggest that SR beta, as an integral membrane protein, mediates the membrane association of SR alpha. The discovery of its guanine nucleotide-binding domain, however, makes it likely that its role is more complex than that of a passive anchor for SR alpha. These findings suggest that a cascade of three directly interacting GTPases functions during protein targeting to the ER membrane.     

The signal recognition particle receptor is a complex that contains two distinct polypeptide chains

Signal recognition particle (SRP) and SRP receptor are known to be essential components of the cellular machinery that targets nascent secretory proteins to the endoplasmic reticulum (ER) membrane. Here we report that the SRP receptor contains, in addition to the previously identified and sequenced 69-kD polypeptide (alpha-subunit, SR alpha), a 30-kD beta-subunit (SR beta). When SRP receptor was purified by SRP-Sepharose affinity chromatography, we observed the co-purification of two other ER membrane proteins. Both proteins are approximately 30 kD in size and are immunologically distinct from each other, as well as from SR alpha and SRP proteins. One of the 30-kD proteins (SR beta) forms a tight complex with SR alpha in detergent solution that is stable to high salt and can be immunoprecipitated with antibodies to either SR alpha or SR beta. Both subunits are present in the ER membrane in equimolar amounts and co-fractionate in constant stoichiometry when rough and smooth liver microsomes are separated on sucrose gradients. We therefore conclude that SR beta is an integral component of SRP receptor. The presence of SR beta was previously masked by proteolytic breakdown products of SR alpha observed by others and by the presence of another 30-kD ER membrane protein (mp30) which co-purifies with SR alpha. Mp30 binds to SRP-Sepharose directly and is present in the ER membrane in several-fold molar excess of SR alpha and SR beta. The affinity of mp30 for SRP suggests that it may serve a yet unknown function in protein translocation.     

Segregation of the signal sequence receptor protein in the rough endoplasmic reticulum membrane

The signal sequence receptor (SSR), an integral membrane glycoprotein of 34 kDa, has previously been shown to be a component of the molecular environment which nascent polypeptide chains meet in passage through the endoplasmic reticulum (ER) membrane. We have used antibodies directed against the SSR and both immunocytochemistry and cell fractionation to determine its distribution in rat liver cells. SSR was found largely restricted to the rough ER. Only small amounts of the protein were detected in smooth ER. These results provide further evidence for a functional differentiation of rough and smooth ER and for a role of SSR in protein translocation across the ER membrane.     

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.     

Early events in preprotein recognition in E. coli: interaction of SRP and trigger factor with nascent polypeptides.

In Escherichia coli, components of a signal recognition particle (SRP) and its receptor have been identified which appear to be essential for efficient translocation of several proteins. In this study we use cross-linking to demonstrate that E. coli SRP interacts with a variety of nascent presecretory proteins and integral inner membrane proteins. Evidence is presented that the interaction is correlated with the hydrophobicity of the core region of the signal sequence and thereby with its ability to promote transport in vivo. A second E. coli component, which is identified as trigger factor, can be efficiently cross-linked to all tested nascent chains derived from both secreted and cytosolic proteins. We propose that SRP and trigger factor act as secretion-specific and general molecular chaperone respectively, early in protein synthesis.     

Molecular dissection of the NH2-terminal signal/anchor sequence of rat dipeptidyl peptidase IV

Dipeptidyl peptidase IV (DPPIV) is a membrane glycoprotein with a type II orientation in the plasma membrane. As shown in a cell-free translation system, the amino-terminal 34 amino acids of rat DPPIV are involved in translocating nascent polypeptide across the membrane of microsomes and in anchoring the translocated polypeptide in the microsomal membrane. The amino-terminal sequence performing this dual function is composed of: a central hydrophobic core of 22 amino acid residues; 6 amino-terminal residues preceding the hydrophobic core (MKTPWK); and 6 residues following the hydrophobic core. The six residues preceding the hydrophobic core are exposed on the outside (cytoplasmic side) of the microsomal membrane. Site-directed mutagenesis studies show that deletion of this cytoplasmic domain, excluding the amino-terminal initiating methionine, does not affect translocation of nascent DPPIV polypeptide, but does affect significantly anchoring of the translocated polypeptide in the microsomal membrane. In contrast, changing the two cytoplasmic Lys to Glu residues or shortening of the hydrophobic core from 22 to 15 residues or converting the last 11e of the shortened hydrophobic core into Ala affects neither translocation across nor anchoring of the DPPIV polypeptide in the microsomal membrane. These and other structural features of the DPPIV amino-terminal signal-anchor sequences are discussed along with other types of sequences for their role in targeting nascent polypeptides to the RER.     

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.     

Signal sequence recognition and targeting of ribosomes to the endoplasmic reticulum by the signal recognition particle do not require GTP.

The identification of GTP-binding sites in the 54-kDa subunit of the signal recognition particle (SRP) and in both the alpha and beta subunits of the SRP receptor has complicated the task of defining the step in the protein translocation reaction that is controlled by the GTP-binding site in the SRP. Ribonucleotide binding assays show that the purified SRP can bind GDP or GTP. However, crosslinking experiments show that SRP54 can recognize the signal sequence of a nascent polypeptide in the absence of GTP. Targeting of SRP-ribosome-nascent polypeptide complexes, formed in the absence of GTP, to microsomal membranes likewise proceeds normally. To separate the GTPase cycles of SRP54 and the alpha subunit of the SRP receptor (SR alpha), we employed an SR alpha mutant that displays a markedly reduced affinity for GTP. We observed that the dissociation of SRP54 from the signal sequence and the insertion of the nascent polypeptide into the translocation site could only occur when GTP binding to SR alpha was permitted. These data suggest that the GTP binding and hydrolysis cycles of both SRP54 and SR alpha are initiated upon formation of the SRP-SRP receptor complex.     

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.     

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.     

The identification of proteins in the proximity of signal-anchor sequences during their targeting to and insertion into the membrane of the ER

Using a photocross-linking approach we have investigated the cytosolic and membrane components involved in the targeting and insertion of signal-anchor proteins into the membrane of the ER. The nascent chains of both type I and type II signal-anchor proteins can be cross-linked to the 54-kD subunit of the signal recognition particle. Upon addition of rough microsomes the type I and type II signal-anchor proteins interact with a number of components. Both types of protein interact with an integral membrane protein, the signal sequence receptor, previously identified by its proximity to preprolactin during its translocation (Wiedmann, M., T.V. Kurzchalia, E. Hartmann, and T.A. Rapoport. 1987. Nature [Lond.] 328:830-833). Three proteins, previously unidentified, were found to be cross-linked to the nascent chains of the signal-anchor proteins. Among them was a 37-kD protein that was found to be the main component interacting with the type I SA protein used. These proteins were not seen in the absence of membranes suggesting they are components of the ER. The ability of the nascent chains to be cross-linked to these identified proteins was shown to be abolished by prior treatment with agents known to disrupt translocation intermediates or ribosomes. We propose that the newly identified proteins function either in the membrane insertion of only a subset of proteins or only at a specific stage of insertion.     

Elongation arrest is not a prerequisite for secretory protein translocation across the microsomal membrane

Signal recognition particle (SRP) is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of small cytoplasmic 7SL RNA. It was previously shown to promote the co-translational translocation of secretory proteins across the endoplasmic reticulum by (a) arresting the elongation of the presecretory nascent chain at a specific point, and (b) interacting with the SRP receptor, an integral membrane protein of the endoplasmic reticulum which is active in releasing the elongation arrest. Recently a procedure was designed by which the particle could be disassembled into its protein and RNA components. We have further separated the SRP proteins into four homogeneous fractions. When recombined with each other and with 7SL RNA, they formed fully active SRP. Particles missing specific proteins were assembled in the hope that some of these would retain some functional activity. SRP(-9/14), the particle lacking the 9-kD and 14-kD polypeptides, was fully active in promoting translocation, but was completely inactive in elongation arrest. This implied that elongation arrest is not a prerequisite for protein translocation. SRP receptor was required for SRP(-9/14)-mediated translocation to occur, and thus must play some role in the translocation process in addition to releasing the elongation arrest.     

The Escherichia coli SRP and SecB targeting pathways converge at the translocon.

Two distinct protein targeting pathways can direct proteins to the Escherichia coli inner membrane. The Sec pathway involves the cytosolic chaperone SecB that binds to the mature region of pre-proteins. SecB targets the pre-protein to SecA that mediates pre-protein translocation through the SecYEG translocon. The SRP pathway is probably used primarily for the targeting and assembly of inner membrane proteins. It involves the signal recognition particle (SRP) that interacts with the hydrophobic targeting signal of nascent proteins. By using a protein cross-linking approach, we demonstrate here that the SRP pathway delivers nascent inner membrane proteins at the membrane. The SRP receptor FtsY, GTP and inner membranes are required for release of the nascent proteins from the SRP. Upon release of the SRP at the membrane, the targeted nascent proteins insert into a translocon that contains at least SecA, SecY and SecG. Hence, as appears to be the case for several other translocation systems, multiple targeting mechanisms deliver a variety of precursor proteins to a common membrane translocation complex of the E.coli inner membrane.