Sec61p and BiP directly facilitate polypeptide translocation into the ER.

Secretory proteins are segregated from cytosolic proteins by their translocation into the endoplasmic reticulum (ER). A modified secretory protein trapped during translocation across the ER membrane can be crosslinked to two previously identified proteins, Sec61p and BiP (Kar2p). The dependence of this cross-linking upon proteins and small molecules was examined. Mutations in SEC62 and SEC63 decrease the ability of Sec61p to be cross-linked to the secretory polypeptide trapped in translocation. ATP is also required for interaction of Sec61p with the secretory protein. Three kar2 alleles display defective translocation in vitro. Two of these alleles also decrease the ability of Sec61p to be cross-linked to the secretory protein. The third allele, while exhibiting a severe translocation defect, does not affect the interaction of Sec61p with the secretory protein. These results suggest that Sec61p is directly involved in translocation and that BiP acts at two stages of the translocation cycle.     

The beta subunit of the Sec61p endoplasmic reticulum translocon interacts with the exocyst complex in Saccharomyces cerevisiae

The exocyst is a conserved protein complex proposed to mediate vesicle tethering at the plasma membrane. Previously, we identified SEB1/SBH1, encoding the beta subunit of the Sec61p ER translocation complex, as a multicopy suppressor of the sec15-1 mutant, defective for one subunit of the exocyst complex. Here we show the functional and physical interaction between components of endoplasmic reticulum translocon and the exocytosis machinery. We show that overexpression of SEB1 suppresses the growth defect in all exocyst sec mutants. In addition, overexpression of SEC61 or SSS1 encoding the other two components of the Sec61p complex suppressed the growth defects of several exocyst mutants. Seb1p was coimmunoprecipitated from yeast cell lysates with Sec15p and Sec8p, components of the exocyst complex, and with Sec4p, a secretory vesicle associated Rab GTPase that binds to Sec15p and is essential for exocytosis. The interaction between Seb1p and Sec15p was abolished in sec15-1 mutant and was restored upon SEB1 overexpression. Furthermore, in wild type cells overexpression of SEB1 as well as SEC4 resulted in increased production of secreted proteins. These findings propose a novel functional and physical link between the endoplasmic reticulum translocation complex and the exocyst.     

Sec72p contributes to the selective recognition of signal peptides by the secretory polypeptide translocation complex

SEC72 encodes the 23-kD subunit of the Sec63p complex, an integral ER membrane protein complex that is required for translocation of presecretory proteins into the ER of Saccharomyces cerevisiae. DNA sequence analysis of SEC72 predicts a 21.6-kD protein with neither a signal peptide nor any transmembrane domains. Antibodies directed against a carboxyl-terminal peptide of Sec72p were used to confirm the membrane location of the protein. SEC72 is not essential for yeast cell growth, although an sec72 null mutant accumulates a subset of secretory precursors in vivo. Experiments using signal peptide chimeric proteins demonstrate that the sec72 translocation defect is associated with the signal peptide rather than with the mature region of the secretory precursor.     

Sec61p is part of the endoplasmic reticulum‐associated degradation machinery

Endoplasmic reticulum‐associated degradation (ERAD) is a cellular pathway for the disposal of misfolded secretory proteins. This process comprises recognition of the misfolded proteins followed by their retro‐translocation across the ER membrane into the cytosol in which polyubiquitination and proteasomal degradation occur. A variety of data imply that the protein import channel Sec61p has a function in the ERAD process. Until now, no physical interactions between Sec61p and other essential components of the ERAD pathway could be found. Here, we establish this link by showing that Hrd3p, which is part of the Hrd‐Der ubiquitin ligase complex, and other core components of the ERAD machinery physically interact with Sec61p. In addition, we study binding of misfolded CPY^* proteins to Sec61p during the process of degradation. We show that interaction with Sec61p is maintained until the misfolded proteins are ubiquitinated on the cytosolic side of the ER. Our observations suggest that Sec61p contacts an ERAD ligase complex for further elimination of ER lumenal misfolded proteins.     

Sec61 complexes form ubiquitous ER Ca2+ leak channels

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis and in calcium signalling. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous pathway for transporting newly synthesized polypeptides into the ER lumen and may also allow calcium leakage from the ER into the cytosol. In this study, planar lipid bilayer experiments demonstrated that the Sec61 complex is permeable to calcium ions. We also investigated whether silencing the SEC61A1 gene affected calcium leakage from the ER. Silencing the SEC61A1 gene using two different siRNAs in HeLa cells for 96 hours had little effect on cell growth and viability. However, calcium leakage from the ER was greatly decreased in the SEC61A1-silenced cells. Thus, the Sec61 complexes that are present in the ER membrane of nucleated cells form calcium leak channels that may play a crucial role in calcium homeostasis.     

Sec61 complexes form ubiquitous ER Ca2+ leak channels

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis and in calcium signalling. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous pathway for transporting newly synthesized polypeptides into the ER lumen and may also allow calcium leakage from the ER into the cytosol. In this study, planar lipid bilayer experiments demonstrated that the Sec61 complex is permeable to calcium ions. We also investigated whether silencing the SEC61A1 gene affected calcium leakage from the ER. Silencing the SEC61A1 gene using two different siRNAs in HeLa cells for 96 hours had little effect on cell growth and viability. However, calcium leakage from the ER was greatly decreased in the SEC61A1-silenced cells. Thus, the Sec61 complexes that are present in the ER membrane of nucleated cells form calcium leak channels that may play a crucial role in calcium homeostasis.     

Sec61p mediates export of a misfolded secretory protein from the endoplasmic reticulum to the cytosol for degradation.

Degradation of misfolded secretory proteins has long been assumed to occur in the lumen of the endoplasmic reticulum (ER). Recent evidence, however, suggests that such proteins are instead degraded by proteasomes in the cytosol, although it remains unclear how the proteins are transported out of the ER. Here we provide the first genetic evidence that Sec61p, the pore-forming subunit of the protein translocation channel in the ER membrane, is directly involved in the export of misfolded secretory proteins. We describe two novel mutants in yeast Sec61p that are cold-sensitive for import into the ER in both intact yeast cells and a cell-free system. Microsomes derived from these mutants are defective in exporting misfolded secretory proteins. These proteins become trapped in the ER and are associated with Sec61p. We conclude that misfolded secretory proteins are exported for degradation from the ER to the cytosol via channels formed by Sec61p.     

Distinct domains within yeast Sec61p involved in post-translational translocation and protein dislocation

The translocation of secretory polypeptides into and across the membrane of the endoplasmic reticulum (ER) occurs at the translocon, a pore-forming structure that orchestrates the transport and maturation of polypeptides at the ER membrane. Recent data also suggest that misfolded or unassembled polypeptides exit the ER via the translocon for degradation by the cytosolic ubiquitin/proteasome pathway. Sec61p is a highly conserved multispanning membrane protein that constitutes a core component of the translocon. We have found that the essential function of the Saccharomyces cerevisiae Sec61p is retained upon deletion of either of two internal regions that include transmembrane domains 2 and 3, respectively. However, a deletion mutation encompassing both of these domains was found to be nonfunctional. Characterization of yeast mutants expressing the viable deletion alleles of Sec61p has revealed defects in post-translational translocation. In addition, the transmembrane domain 3 deletion mutant is induced for the unfolded protein response and is defective in the dislocation of a misfolded ER protein. These data demonstrate that the various activities of Sec61p can be functionally dissected. In particular, the transmembrane domain 2 region plays a role in post-translational translocation that is required neither for cotranslational translocation nor for protein dislocation.     

Stability and function of the Sec61 translocation complex depends on the Sss1p tail-anchor sequence

Sss1p, an essential component of the heterotrimeric Sec61 complex in the ER (endoplasmic reticulum), is a tail-anchored protein whose precise mechanism of action is largely unknown. Tail-anchored proteins are involved in many cellular processes and are characterized by a single transmembrane sequence at or near the C-terminus. The Sec61 complex is the molecular machine through which secretory and membrane proteins translocate into and across the ER membrane. To understand the function of the tail anchor of Sss1p, we introduced mutations into the tail-anchor sequence and analysed the resulting yeast phenotypes. Point mutations in the C-terminal hydrophobic core of the tail anchor of Sss1p were identified that allowed Sss1p assembly into Sec61 complexes, but resulted in diminished growth, defects in co- and post-translational translocation, inefficient ribosome binding to Sec61 complexes, reduction in the stability of both heterotrimeric Sec61 and heptameric Sec complexes and a complete breakdown of ER structure. The underlying defect caused by the mutations involves loss of a stabilizing function of the Sss1p tail-anchor sequence for both the heterotrimeric Sec61 and the heptameric Sec complexes. These results indicate that by stabilizing multiprotein membrane complexes, the hydrophobic core of a tail-anchor sequence can be more than a simple membrane anchor.     

Sec61p-independent degradation of the tail-anchored ER membrane protein Ubc6p.

Tail-anchored proteins are distinct from other membrane proteins as they are thought to insert into the endoplasmic reticulum (ER) membrane independently of Sec61p translocation pores. These pores not only mediate import but are also assumed to catalyze export of proteins in a process called ER-associated protein degradation (ERAD). In order to examine the Sec61p dependence of the export of tail-anchored proteins, we analyzed the degradation pathway of a tail-anchored ER membrane protein, the ubiquitin-conjugating enzyme 6 (Ubc6p). In contrast to other ubiquitin conjugating enzymes (Ubcs), Ubc6p is naturally short-lived. Its proteolysis is mediated specifically by the unique Ubc6p tail region. Degradation further requires the activity of Cue1p-assembled Ubc7p, and its own catalytic site cysteine. However, it occurs independently of the other ERAD components Ubc1p, Hrd1p/Der3p, Hrd3p and Der1p. In contrast to other natural ERAD substrates, proteasomal mutants accumulate a membrane-bound degradation intermediate of Ubc6p. Most interestingly, mutations in SEC61 do not reduce the turnover of full-length Ubc6p nor cause a detectable accumulation of degradation intermediates. These data are in accordance with a model in which tail-anchored proteins can be extracted from membranes independently of Sec61p.     

High-Copy Suppressor Analysis Reveals a Physical Interaction between Sec34p and Sec35p, a Protein Implicated in Vesicle Docking

A temperature-sensitive mutant, sec34-2, is defective in the late stages of endoplasmic reticulum (ER)-to-Golgi transport. A high-copy suppressor screen that uses the sec34-2 mutant has resulted in the identification of the SEC34 structural gene and a novel gene called GRP1. GRP1 encodes a previously unidentified hydrophilic yeast protein related to the mammalian Golgi protein golgin-160. Although GRP1 is not essential for growth, the grp1Delta mutation displays synthetic lethal interactions with several mutations that result in ER accumulation and a block in the late stages of ER-to-Golgi transport, but not with those that block the budding of vesicles from the ER. Our findings suggest that Grp1p may facilitate membrane traffic indirectly, possibly by maintaining Golgi function. In an effort to identify genes whose products physically interact with Sec34p, we also tested the ability of overexpressed SEC34 to suppress known secretory mutations that block vesicular traffic between the ER and the Golgi. This screen revealed that SEC34 specifically suppresses sec35-1. SEC34 encodes a hydrophilic protein of approximately 100 kDa. Like Sec35p, which has been implicated in the tethering of ER-derived vesicles to the Golgi, Sec34p is predominantly soluble. Sec34p and Sec35p stably associate with each other to form a multiprotein complex of approximately 480 kDa. These data indicate that Sec34p acts in conjunction with Sec35p to mediate a common step in vesicular traffic.     

Cancer associated mutations in Sec61γ alter the permeability of the ER translocase

Translocation of secretory and integral membrane proteins across or into the ER membrane occurs via the Sec61 complex, a heterotrimeric protein complex possessing two essential sub-units, Sec61p/Sec61α and Sss1p/Sec61γ and the non-essential Sbh1p/Sec61β subunit. In addition to forming a protein conducting channel, the Sec61 complex maintains the ER permeability barrier, preventing flow of molecules and ions. Loss of Sec61 integrity is detrimental and implicated in the progression of disease. The Sss1p/Sec61γ C-terminus is juxtaposed to the key gating module of Sec61p/Sec61α and is important for gating the translocon. Inspection of the cancer genome database identifies six mutations in highly conserved amino acids of Sec61γ/Sss1p. We identify that five out of the six mutations identified affect gating of the ER translocon, albeit with varying strength. Together, we find that mutations in Sec61γ that arise in malignant cells result in altered translocon gating dynamics, this offers the potential for the translocon to represent a target in co-therapy for cancer treatment.     

Sec3p is needed for the spatial regulation of secretion and for the inheritance of the cortical endoplasmic reticulum

Sec3p is a component of the exocyst complex that tethers secretory vesicles to the plasma membrane at exocytic sites in preparation for fusion. Unlike all other exocyst structural genes, SEC3 is not essential for growth. Cells lacking Sec3p grow and secrete surprisingly well at 25 degrees C; however, late markers of secretion, such as the vesicle marker Sec4p and the exocyst subunit Sec8p, localize more diffusely within the bud. Furthermore, sec3Delta cells are strikingly round relative to wild-type cells and are unable to form pointed mating projections in response to alpha factor. These phenotypes support the proposed role of Sec3p as a spatial landmark for secretion. We also find that cells lacking Sec3p exhibit a dramatic defect in the inheritance of cortical ER into the bud, whereas the inheritance of mitochondria and Golgi is unaffected. Overexpression of Sec3p results in a prominent patch of the endoplasmic reticulum (ER) marker Sec61p-GFP at the bud tip. Cortical ER inheritance in yeast has been suggested to involve the capture of ER tubules at the bud tip. Sec3p may act in this process as a spatial landmark for cortical ER inheritance.     

Structural and functional dissection of Sec62p, a membrane-bound component of the yeast endoplasmic reticulum protein import machinery.

SEC62 is required for the import of secretory protein precursors into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae. The DNA sequence of SEC62 predicts a 32-kDa polypeptide with two potential membrane-spanning segments. Two antisera directed against different portions of the SEC62 coding region specifically detected a 30-kDa polypeptide in cell extracts. A combination of subcellular fractionation, detergent and alkali extraction, and indirect immunofluorescence studies indicated that Sec62p is intimately associated with the ER membrane. Protease digestion of intact microsomes and analysis of the oligosaccharide content of a set of Sec62p-invertase hybrid proteins suggested that Sec62p spans the ER membrane twice, displaying hydrophilic amino- and carboxy-terminal domains towards the cytosol. Sec62p-invertase hybrid proteins that lack the Sec62p C terminus failed to complement the sec62-l mutation and dramatically inhibited the growth of sec62-l cells at a normally permissive temperature. The inhibitory action of toxic Sec62p-invertase hybrids was partially counteracted by the overexpression of Sec63p. Taken together, these data suggest that the C-terminal domain of Sec62p performs an essential function and that the N-terminal domain associates with other components of the translocation machinery, including Sec63p.     

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.     

The Saccharomyces cerevisiae SEC20 gene encodes a membrane glycoprotein which is sorted by the HDEL retrieval system.

The SEC20 gene product (Sec20p) is required for endoplasmic reticulum (ER) to Golgi transport in the yeast secretory pathway. We have cloned the SEC20 gene by complementation of the temperature sensitive phenotype of a sec20-1 strain. The DNA sequence predicts a 44 kDa protein with a single membrane-spanning region; Sec20p has an apparent molecular weight of 50 kDa and behaves as an integral membrane protein with carbohydrate modifications that appear to be O-linked. A striking feature of this protein is its C-terminal sequence, which consists of the tetrapeptide HDEL. This signal is known to be required for the retrieval of soluble ER proteins from early Golgi compartments, but has not previously been observed on a membrane protein. The HDEL sequence of Sec20p is not essential for viability but helps to maintain intracellular levels of the protein. Depletion of Sec20p from cells results in the accumulation of an extensive network of ER and clusters of small vesicles. We suggest a possible role for the SEC20 product in the targeting of transport vesicles to the Golgi apparatus.     

Binding of ribosomes to the rough endoplasmic reticulum mediated by the Sec61p-complex

The cotranslational translocation of proteins across the ER membrane involves the tight binding of translating ribosomes to the membrane, presumably to ribosome receptors. The identity of the latter has been controversial. One putative receptor candidate is Sec61 alpha, a multi- spanning membrane protein that is associated with two additional membrane proteins (Sec61 beta and gamma) to form the Sec61p-complex. Other receptors of 34 and 180 kD have also been proposed on the basis of their ability to bind at low salt concentration ribosomes lacking nascent chains. We now show that the Sec61p-complex has also binding activity but that, at low salt conditions, it accounts for only one third of the total binding sites in proteoliposomes reconstituted from a detergent extract of ER membranes. Under these conditions, the assay has also limited specificity with respect to ribosomes. However, if the ribosome-binding assay is performed at physiological salt concentration, most of the unspecific binding is lost; the Sec61p- complex then accounts for the majority of specific ribosome-binding sites in reconstituted ER membranes. To study the membrane interaction of ribosomes participating in protein translocation, native rough microsomes were treated with proteases. The amount of membrane-bound ribosomes is only slightly reduced by protease treatment, consistent with the protease-resistance of Sec61 alpha which is shielded by these ribosomes. In contrast, p34 and p180 can be readily degraded, indicating that they are not essential for the membrane anchoring of ribosomes in protease-treated microsomes. These data provide further evidence that the Sec61p-complex is responsible for the membrane- anchoring of ribosomes during translocation and make it unlikely that p34 or p180 are essential for this process.     

The engagement of Sec61p in the ER dislocation process

Sec61p comprises the endoplasmic reticulum (ER) channel through which nascent polypeptides are imported and from which malfolded proteins have been suggested to be exported, or dislocated, back to the cytoplasm. We have devised a genetic screen for dislocation-specific mutant alleles of SEC61 from S. cerevisiae by employing the unfolded protein response to report on the accumulation of misfolded proteins in the ER. Three of the isolated sec61 alleles are fully proficient in protein translocation into the ER, but defective in the elimination of a misfolded ER luminal substrate and a short-lived ER membrane-spanning model protein, which are otherwise rapidly degraded by cytoplasmic proteolysis in wild-type cells. Our results point to the fourth luminal loop and third transmembrane domain of Sec61p that markedly influence dislocation. We suggest that distinct features of the Sec61-translocon direct the two-way translocation processes.     

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.