Sec61p is the main ribosome receptor in the endoplasmic reticulum of Saccharomyces cerevisiae

A characteristic feature of the co-translational protein translocation into the endoplasmic reticulum (ER) is the tight association of the translating ribosomes with the translocation sites in the membrane. Biochemical analyses identified the Sec61 complex as the main ribosome receptor in the ER of mammalian cells. Similar experiments using purified homologues from the yeast Saccharomyces cerevisiae, the Sec61p complex and the Ssh1p complex, respectively, demonstrated that they bind ribosomes with an affinity similar to that of the mammalian Sec61 complex. However, these studies did not exclude the presence of other proteins that may form abundant ribosome binding sites in the yeast ER. We now show here that similar to the situation found in mammals in the yeast Saccharomyces cerevisiae the two Sec61-homologues Sec61p and Ssh1p are essential for the formation of high-affinity ribosome binding sites in the ER membrane. The number of binding sites formed by Ssh1p under standard growth conditions is at least 4 times less than those formed by Sec61p.     

Interactions between Sec complex and prepro-alpha-factor during posttranslational protein transport into the endoplasmic reticulum

Posttranslational translocation of prepro-alpha-factor (ppalphaF) across the yeast endoplasmic reticulum membrane begins with the binding of the signal sequence to the Sec complex, a membrane component consisting of the trimeric Sec61p complex and the tetrameric Sec62p/63p complex. We show by photo-cross-linking that the signal sequence is bound directly to a site where it contacts simultaneously Sec61p and Sec62p, suggesting that there is a single signal sequence recognition step. We found no evidence for the simultaneous contact of the signal sequence with two Sec61p molecules. To identify transmembrane segments of Sec61p that line the actual translocation pore, a late translocation intermediate of ppalphaF was generated with photoreactive probes incorporated into the mature portion of the polypeptide. Cross-linking to multiple regions of Sec61p was observed. In contrast to the signal sequence, neighboring positions of the mature portion of ppalphaF had similar interactions with Sec61p. These data suggest that the channel pore is lined by several transmembrane segments, which have no significant affinity for the translocating polypeptide chain.     

Architecture of the active post‐translational Sec translocon

In eukaryotes, most secretory and membrane proteins are targeted by an N‐terminal signal sequence to the endoplasmic reticulum, where the trimeric Sec61 complex serves as protein‐conducting channel (PCC). In the post‐translational mode, fully synthesized proteins are recognized by a specialized channel additionally containing the Sec62, Sec63, Sec71, and Sec72 subunits. Recent structures of this Sec complex in the idle state revealed the overall architecture in a pre‐opened state. Here, we present a cryo‐EM structure of the yeast Sec complex bound to a substrate, and a crystal structure of the Sec62 cytosolic domain. The signal sequence is inserted into the lateral gate of Sec61α similar to previous structures, yet, with the gate adopting an even more open conformation. The signal sequence is flanked by two Sec62 transmembrane helices, the cytoplasmic N‐terminal domain of Sec62 is more rigidly positioned, and the plug domain is relocated. We crystallized the Sec62 domain and mapped its interaction with the C‐terminus of Sec63. Together, we obtained a near‐complete and integrated model of the active Sec complex.     

Mammalian Sec61 is associated with Sec62 and Sec63

In yeast, efficient protein transport across the endoplasmic reticulum (ER) membrane may occur co-translationally or post-translationally. The latter process is mediated by a membrane protein complex that consists of the Sec61p complex and the Sec62p-Sec63p subcomplex. In contrast, in mammalian cells protein translocation is almost exclusively co-translational. This transport depends on the Sec61 complex, which is homologous to the yeast Sec61p complex and has been identified in mammals as a ribosome-bound pore-forming membrane protein complex. We report here the existence of ribosome-free mammalian Sec61 complexes that associate with two ubiquitous proteins of the ER membrane. According to primary sequence analysis both proteins display homology to the yeast proteins Sec62p and Sec63p and are therefore named Sec62 and Sec63, respectively. The probable function of the mammalian Sec61-Sec62-Sec63 complex is discussed with respect to its abundance in ER membranes, which, in contrast to yeast ER membranes, apparently lack efficient post-translational translocation activity.     

Evolutionarily conserved binding of ribosomes to the translocation channel via the large ribosomal RNA

During early stages of cotranslational protein translocation across the endoplasmic reticulum (ER) membrane the ribosome is targeted to the heterotrimeric Sec61p complex, the major component of the protein-conducting channel. We demonstrate that this interaction is mediated by the 28S rRNA of the eukaryotic large ribosomal subunit. Bacterial ribosomes also bind via their 23S rRNA to the bacterial homolog of the Sec61p complex, the SecYEG complex. Eukaryotic ribosomes bind to the SecYEG complex, and prokaryotic ribosomes to the Sec61p complex. These data indicate that rRNA-mediated interaction of ribosomes with the translocation channel occurred early in evolution and has been conserved.     

Identification of cytoplasmic residues of Sec61p involved in ribosome binding and cotranslational translocation

The cytoplasmic surface of Sec61p is the binding site for the ribosome and has been proposed to interact with the signal recognition particle receptor during targeting of the ribosome nascent chain complex to the translocation channel. Point mutations in cytoplasmic loops six (L6) and eight (L8) of yeast Sec61p cause reductions in growth rates and defects in the translocation of nascent polypeptides that use the cotranslational translocation pathway. Sec61 heterotrimers isolated from the L8 sec61 mutants have a greatly reduced affinity for 80S ribosomes. Cytoplasmic accumulation of protein precursors demonstrates that the initial contact between the large ribosomal subunit and the Sec61 complex is important for efficient insertion of a nascent polypeptide into the translocation pore. In contrast, point mutations in L6 of Sec61p inhibit cotranslational translocation without significantly reducing the ribosome-binding activity, indicating that the L6 and L8 sec61 mutants affect different steps in the cotranslational translocation pathway.     

Yeast ribosomes bind to highly purified reconstituted Sec61p complex and to mammalian p180

To determine whether the yeast Sec61p translocation pore is a high-affinity ribosome receptor in the endoplasmic reticulum, we isolated the Sec61p complex using an improved protocol in which contaminants found previously to be associated with the complex are absent. The purified complex, which contains Sec61p with an amino terminal hexahistidine tag, was active since it rescued a sec61–3 post-translational translocation defect in a reconstituted system. Co-reconstitution of the Sec61p and Sec63p complexes into liposomes failed to support post-translational translocation, suggesting that Sec62p is required for this process. By Scatchard analysis, the purified Sec61p complex bound to yeast ribosomes when reconstituted into liposomes with a K_D of 5.6 n m , a value similar to the K_D obtained when ribosome binding to total microsomal protein was measured (2.7 n m ). In addition, a mammalian protein, p180, which has been proposed to be a ribosome receptor, was expressed in yeast, and endoplasmic reticulum-derived microsomes isolated from this strain exhibited ∼2.3-fold greater binding to yeast ribosomes. Despite this increase in ribosome binding, neither co- nor post-translational translocation was compromised in vivo . In sum, our data suggest that the Sec61p complex is a ribosome receptor in the yeast endoplasmic reticulum membrane.     

Sec61β facilitates the maintenance of endoplasmic reticulum homeostasis by associating microtubules

Sec61β, a subunit of the Sec61 translocon complex, is not essential in yeast and commonly used as a marker of endoplasmic reticulum (ER). In higher eukaryotes, such as Drosophila, deletion of Sec61β causes lethality, but its physiological role is unclear. Here, we show that Sec61β interacts directly with microtubules. Overexpression of Sec61β containing small epitope tags, but not a RFP tag, induces dramatic bundling of the ER and microtubule. A basic region in the cytosolic domain of Sec61β is critical for microtubule association. Depletion of Sec61β induces ER stress in both mammalian cells and Caenorhabditis elegans, and subsequent restoration of ER homeostasis correlates with the microtubule binding ability of Sec61β. Loss of Sec61β causes increased mobility of translocon complexes and reduced level of membrane-bound ribosomes. These results suggest that Sec61β may stabilize protein translocation by linking translocon complex to microtubule and provide insight into the physiological function of ER-microtubule interaction.     

Molecular architecture of the ER translocase probed by chemical crosslinking of Sss1p to complementary fragments of Sec61p.

The heterotrimeric Sec61p complex is a key component of the protein translocation apparatus of the endoplasmic reticulum membrane. The complex characterized from yeast includes Sec61p, a 10-transmembrane-domain membrane protein which has a direct interaction with Sss1p, a small C-terminal anchor protein. In order to gain some insight into the architecture of this complex we have functionally expressed Sec61p as complementary N- and C-terminal fragments. Chemical crosslinking of Sss1p to specific Sec61p fragments in these functional combinations and suppression of sec61 mutants by over-expression of Sss1p have led to identification of the region which includes transmembrane domains TM6, TM7 and TM8 (amino acid residues L232-R406) of Sec61p as a major site of interaction with Sss1p.     

A second trimeric complex containing homologs of the Sec61p complex functions in protein transport across the ER membrane of S. cerevisiae.

Yeast microsomes contain a heptameric Sec complex involved in post-translational protein transport that is composed of a heterotrimeric Sec61p complex and a tetrameric Sec62-Sec63 complex. The trimeric Sec61p complex also exists as a separate entity that probably functions in co-translational protein transport, like its homolog in mammals. We have now discovered in the yeast endoplasmic reticulum membrane a second, structurally related trimeric complex, named Ssh1p complex. It consists of Ssh1p1 (Sec sixty-one homolog 1), a rather distant relative of Sec61p, of Sbh2p, a homolog of the Sbh1p subunit of the Sec61p complex, and of Sss1p, a component common to both trimeric complexes. In contrast to Sec61p, Ssh1p is not essential for cell viability but it is required for normal growth rates. Sbh1p and Sbh2p individually are also not essential, but cells lacking both proteins are impaired in their growth at elevated temperatures and accumulate precursors of secretory proteins; microsomes isolated from these cells also exhibit a reduced rate of post-translational protein transport. Like the Sec61p complex, the Ssh1p complex interacts with membrane-bound ribosomes, but it does not associate with the Sec62-Sec63p complex to form a heptameric Sec complex. We therefore propose that it functions exclusively in the co-translational pathway of protein transport.     

The β Subunit of the Sec61 Complex Facilitates Cotranslational Protein Transport and Interacts with the Signal Peptidase during Translocation

The Sec61 complex is the central component of the protein translocation apparatus of the ER membrane. We have addressed the role of the β subunit (Sec61β) during cotranslational protein translocation. With a reconstituted system, we show that a Sec61 complex lacking Sec61β is essentially inactive when elongation and membrane targeting of a nascent chain occur at the same time. The translocation process is perturbed at a step where the nascent chain would be inserted into the translocation channel. However, if sufficient time is given for the interaction of the nascent polypeptide with the mutant Sec61 complex, translocation is almost normal. Thus Sec61β kinetically facilitates cotranslational translocation, but is not essential for it.

Using chemical cross-linking we show that Sec61β not only interacts with subunits of the Sec61 complex but also with the 25-kD subunit of the signal peptidase complex (SPC25), thus demonstrating for the first time a tight interaction between the SPC and the Sec61 complex. Interestingly, the cross-links between Sec61β and SPC25 and between Sec61β and Sec61α depend on the presence of membrane-bound ribosomes, suggesting that these interactions are induced when translocation is initiated. We propose that the SPC is transiently recruited to the translocation site, thus enhancing its activity.

     

The protein translocation apparatus of the rough endoplasmic reticulum, its associated proteins, and the mechanism of translocation.

The demonstration that the yeast Sec61, Sec62, and Sec63 proteins are assembled into a multisubunit complex in the yeast endoplasmic reticulum was of particular significance in a year when protein, and nucleic-sequence analyses revealed interesting homologies between pathways of protein transport in mammals and yeast, and possibly in Escherichia coli.     

Interaction of Pseudomonas aeruginosa Exotoxin A with the human Sec61 complex suppresses passive calcium efflux from the endoplasmic reticulum

According to live-cell calcium-imaging experiments, the Sec61 complex is a passive calcium-leak channel in the human endoplasmic reticulum (ER) membrane that is regulated by ER luminal immunoglobulin heavy chain binding protein (BiP) and cytosolic Ca²⁺-calmodulin. In single channel measurements, the open Sec61 complex is Ca²⁺ permeable. It can be closed not only by interaction with BiP or Ca²⁺-calmodulin, but also with Pseudomonas aeruginosa Exotoxin A which can enter human cells by retrograde transport. Exotoxin A has been shown to interact with the Sec61 complex and, thereby, inhibit ER export of immunogenic peptides into the cytosol. Here, we show that Exotoxin A also inhibits passive Ca²⁺ leakage from the ER in human cells, and we characterized the N-terminus of the Sec61 α-subunit as the relevant binding site for Exotoxin A.     

Role of Sec61alpha in the regulated transfer of the ribosome-nascent chain complex from the signal recognition particle to the translocation channel

Targeting of ribosome-nascent chain complexes to the translocon in the endoplasmic reticulum is mediated by the concerted action of the signal recognition particle (SRP) and the SRP receptor (SR). Ribosome-stripped microsomes were digested with proteases to sever cytoplasmic domains of SRalpha, SRbeta, TRAM, and the Sec61 complex. We characterized protein translocation intermediates that accumulate when Sec61alpha or SRbeta is inactivated by proteolysis. In the absence of a functional Sec61 complex, dissociation of SRP54 from the signal sequence is blocked. Experiments using SR proteoliposomes confirmed the assembly of a membrane-bound posttargeting intermediate. These results strongly suggest that the Sec61 complex regulates the GTP hydrolysis cycle of the SRP-SR complex at the stage of signal sequence dissociation from SRP54.     

Interaction of Pseudomonas aeruginosa Exotoxin A with the human Sec61 complex suppresses passive calcium efflux from the endoplasmic reticulum

According to live-cell calcium-imaging experiments, the Sec61 complex is a passive calcium-leak channel in the human endoplasmic reticulum (ER) membrane that is regulated by ER luminal immunoglobulin heavy chain binding protein (BiP) and cytosolic Ca²⁺-calmodulin. In single channel measurements, the open Sec61 complex is Ca²⁺ permeable. It can be closed not only by interaction with BiP or Ca²⁺-calmodulin, but also with Pseudomonas aeruginosa Exotoxin A which can enter human cells by retrograde transport. Exotoxin A has been shown to interact with the Sec61 complex and, thereby, inhibit ER export of immunogenic peptides into the cytosol. Here, we show that Exotoxin A also inhibits passive Ca²⁺ leakage from the ER in human cells, and we characterized the N-terminus of the Sec61 α-subunit as the relevant binding site for Exotoxin A.     

Signal Recognition Particle-dependent Targeting of Ribosomes to the Rough Endoplasmic Reticulum in the Absence and Presence of the Nascent Polypeptide-associated Complex

Proteins with RER-specific signal sequences are cotranslationally translocated across the rough endoplasmic reticulum through a proteinaceous channel composed of oligomers of the Sec61 complex. The Sec61 complex also binds ribosomes with high affinity. The dual function of the Sec61 complex necessitates a mechanism to prevent signal sequence-independent binding of ribosomes to the translocation channel. We have examined the hypothesis that the signal recognition particle (SRP) and the nascent polypeptide-associated complex (NAC), respectively, act as positive and negative regulatory factors to mediate the signal sequence-specific attachment of the ribosome-nascent chain complex (RNC) to the translocation channel. Here, SRP-independent translocation of a nascent secretory polypeptide was shown to occur in the presence of endogenous wheat germ or rabbit reticulocyte NAC. Furthermore, SRP markedly enhanced RNC binding to the translocation channel irrespective of the presence of NAC. Binding of RNCs, but not SRP-RNCs, to the Sec61 complex is competitively inhibited by 80S ribosomes. Thus, the SRP-dependent targeting pathway provides a mechanism for delivery of RNCs to the translocation channel that is not inhibited by the nonselective interaction between the ribosome and the Sec61 complex.     

Role of the cytoplasmic segments of Sec61alpha in the ribosome-binding and translocation-promoting activities of the Sec61 complex

The Sec61 complex performs a dual function in protein translocation across the RER, serving as both the high affinity ribosome receptor and the translocation channel. To define regions of the Sec61 complex that are involved in ribosome binding and translocation promotion, ribosome-stripped microsomes were subjected to limited digestions using proteases with different cleavage specificities. Protein immunoblot analysis using antibodies specific for the NH(2) and COOH terminus of Sec61alpha was used to map the location of proteolysis cleavage sites. We observed a striking correlation between the loss of binding activity for nontranslating ribosomes and the digestion of the COOH- terminal tail or cytoplasmic loop 8 of Sec61alpha. The proteolyzed microsomes were assayed for SRP-independent translocation activity to determine whether high affinity binding of the ribosome to the Sec61 complex is a prerequisite for nascent chain transport. Microsomes that do not bind nontranslating ribosomes at physiological ionic strength remain active in SRP-independent translocation, indicating that the ribosome binding and translocation promotion activities of the Sec61 complex do not strictly correlate. Translocation-promoting activity was most severely inhibited by cleavage of cytosolic loop 6, indicating that this segment is a critical determinant for this function of the Sec61 complex.     

The Brl domain in Sec63p is required for assembly of functional endoplasmic reticulum translocons

Protein translocation into the endoplasmic reticulum occurs at pore-forming structures known as translocons. In yeast, two different targeting pathways converge at a translocation pore formed by the Sec61 complex. The signal recognition particle-dependent pathway targets nascent precursors co-translationally, whereas the Sec62p-dependent pathway targets polypeptides post-translationally. In addition to the Sec61 complex, both pathways also require Sec63p, an integral membrane protein of the Hsp40 family, and Kar2p, a soluble Hsp70 located in the ER lumen. Using a series of mutant alleles, we demonstrate that a conserved Brl (Brr2-like) domain in the COOH-terminal cytosolic region of Sec63p is essential for function both in vivo and in vitro. We further demonstrate that this domain is required for assembly of two oligomeric complexes of 350 and 380 kDa, respectively. The larger of these corresponds to the heptameric "SEC complex" required for post-translational translocation. However, the 350-kDa complex represents a newly defined hexameric SEC' complex comprising Sec61p, Sss1p, Sbh1p, Sec63p, Sec71p, and Sec72p. Our data indicate that the SEC' complex is required for co-translational protein translocation across the yeast ER membrane.     

Identification of a Novel Stage of Ribosome/Nascent Chain Association with the Endoplasmic Reticulum Membrane

Protein translocation in the mammalian endoplasmic reticulum (ER) occurs cotranslationally and requires the binding of translationally active ribosomes to components of the ER membrane. Three candidate ribosome receptors, p180, p34, and Sec61p, have been identified in binding studies with inactive ribosomes, suggesting that ribosome binding is mediated through a receptor-ligand interaction. To determine if the binding of nascent chain-bearing ribosomes is regulated in a manner similar to inactive ribosomes, we have investigated the ribosome/nascent chain binding event that accompanies targeting. In agreement with previous reports, indicating that Sec61p displays the majority of the ER ribosome binding activity, we observed that Sec61p is shielded from proteolytic digestion by native, bound ribosomes. The binding of active, nascent chain bearing ribosomes to the ER membrane is, however, insensitive to the ribosome occupancy state of Sec61p. To determine if additional, Sec61p independent, stages of the ribosome binding reaction could be identified, ribosome/nascent chain binding was assayed as a function of RM concentration. At limiting RM concentrations, a protease resistant ribosome-membrane junction was formed, yet the nascent chain was salt extractable and cross-linked to Sec61p with low efficiency. At nonlimiting RM concentrations, bound nascent chains were protease and salt resistant and cross-linked to Sec61p with higher efficiency. On the basis of these and other data, we propose that ribosome binding to the ER membrane is a multi-stage process comprised of an initial, Sec61p independent binding event, which precedes association of the ribosome/nascent chain complex with Sec61p.     

The beta-subunit of the protein-conducting channel of the endoplasmic reticulum functions as the guanine nucleotide exchange factor for the beta-subunit of the signal recognition particle receptor

Cotranslational protein transport to the endoplasmic reticulum is controlled by the concerted interaction of three GTPases: the SRP54 subunit of the signal recognition particle (SRP) and the alpha- and beta-subunits of the SRP receptor (SR). SRbeta is related to ADP-ribosylation factor (ARF)-type GTPases, and the recently published crystal structure of SRbeta-GTP in complex with the binding domain of SRalpha suggested that SRbeta, like all ARF-type GT-Pases, requires a guanine nucleotide exchange factor (GEF) for function. Searching the sequence data base, we identified significant sequence similarity between the Sec7 domain of ARF-GEFs and the cytosolic domains of the beta-subunits of the two homologous heterotrimeric protein-conducting channels in yeast. Using a fluorescence nucleotide exchange assay, we show that the beta-subunits of the heterotrimeric protein-conducting channels function as the GEFs for SRbeta. Both the cytosolic domain of Sec61beta as well as the holo-Sec61beta, when part of the isolated trimeric Sec61p complex, function as the GEF for SRbeta, whereas the same Sec61beta, when part of the heptameric complex that facilitates posttranslational protein transport, is inactive as the GEF for SRbeta