The ER Protein Translocation Channel Subunit Sbh1 Controls Virulence of Cryptococcus neoformans.

The fungal pathogen Cryptococcus neoformans is distinguished by a cell-wall-anchored polysaccharide capsule that is critical for virulence. Biogenesis of both cell wall and capsule relies on the secretory pathway. Protein secretion begins with polypeptide translocation across the endoplasmic reticulum (ER) membrane through a highly conserved channel formed by three proteins: Sec61, Sbh1, and Sss1. Sbh1, the most divergent, contains multiple phosphorylation sites, which may allow it to regulate entry into the secretory pathway in a species- and protein-specific manner. Absence of SBH1 causes a cell-wall defect in both Saccharomyces cerevisiae and C. neoformans, although other phenotypes differ. Notably, proteomic analysis showed that when cryptococci are grown in conditions that mimic aspects of the mammalian host environment (tissue culture medium, 37°C, 5% CO2), a set of secretory and transmembrane proteins is upregulated in wild-type, but not in Δsbh1 mutant cells. The Sbh1-dependent proteins show specific features of their ER targeting sequences that likely cause them to transit less efficiently into the secretory pathway. Many also act in cell-wall biogenesis, while several are known virulence factors. Consistent with these observations, the C. neoformans Δsbh1 mutant is avirulent in a mouse infection model. We conclude that, in the context of conditions encountered during infection, Sbh1 controls the entry of virulence factors into the secretory pathway of C. neoformans, and thereby regulates fungal pathogenicity. IMPORTANCE Cryptococcus neoformans is a yeast that causes almost 200,000 deaths worldwide each year, mainly of immunocompromised individuals. The surface structures of this pathogen, a protective cell wall surrounded by a polysaccharide capsule, are made and maintained by proteins that are synthesized inside the cell and travel outwards through the secretory pathway. A protein called Sbh1 is part of the machinery that determines which polypeptides enter this export pathway. We found that when Sbh1 is absent, both C. neoformans and the model yeast S. cerevisiae show cell-wall defects. Lack of Sbh1 also changes the pattern of secretion of both transmembrane and soluble proteins, in a manner that depends on characteristics of their sequences. Notably, multiple proteins that are normally upregulated in conditions similar to those encountered during infection, including several needed for cryptococcal virulence, are no longer increased. Sbh1 thereby regulates the ability of this important pathogen to cause disease.

Sec61 channel subunit Sbh1/Sec61ß promotes ER translocation of proteins with suboptimal targeting sequences and is fine-tuned by phosphorylation.

The highly conserved endoplasmic reticulum (ER) protein translocation channel contains one nonessential subunit, Sec61ß/Sbh1, whose function is poorly understood so far. Its intrinsically unstructured cytosolic domain makes transient contact with ER-targeting sequences in the cytosolic channel vestibule and contains multiple phosphorylation sites suggesting a potential for regulating ER protein import. In a microscopic screen, we show that 12% of a GFP-tagged secretory protein library depends on Sbh1 for translocation into the ER. Sbh1-dependent proteins had targeting sequences with less pronounced hydrophobicity and often no charge bias or an inverse charge bias which reduces their insertion efficiency into the Sec61 channel. We determined that mutating two N-terminal, proline-flanked phosphorylation sites in the Sbh1 cytosolic domain to alanine phenocopied the temperature-sensitivity of a yeast strain lacking SBH1 and its ortholog SBH2. The phosphorylation site mutations reduced translocation into the ER of a subset of Sbh1-dependent proteins, including enzymes whose concentration in the ER lumen is critical for ER proteostasis. In addition, we found that ER import of these proteins depended on the activity of the phospho-S/T-specific proline isomerase Ess1 (PIN1 in mammals). We conclude that Sbh1 promotes ER translocation of substrates with suboptimal targeting sequences and that its activity can be regulated by a conformational change induced by N-terminal phosphorylation.

Effect of Sec62 on the conformation of the Sec61 channel in yeast.

Most eukaryotic secretory and membrane proteins are funneled by the Sec61 complex into the secretory pathway. Furthermore, some substrate peptides rely on two essential accessory proteins, Sec62 and Sec63, being present to assist with their translocation via the Sec61 channel in post-translational translocation. Cryo-electron microscopy (cryo-EM) recently succeeded in determining atomistic structures of unbound and signal sequence-engaged Sec complexes from Saccharomyces cerevisiae, involving the Sec61 channel and the proteins Sec62, Sec63, Sec71 and Sec72. In this study, we investigated the conformational effects of Sec62 on Sec61. Indeed, we observed in molecular dynamics simulations that the conformational dynamics of lateral gate, plug and pore region of Sec61 are altered by the presence/absence of Sec62. In molecular dynamics simulations that were started from the cryo-EM structures of Sec61 coordinated to Sec62 or of apo Sec61, we observed that the luminal side of the lateral gate gradually adopts a closed conformation similar to the apo state during unbound state simulations. In contrast, it adopts a wider conformation in the bound state. Furthermore, we demonstrate that the conformation of the active (substrate-bound) state of the Sec61 channel shifts toward an alternative conformation in the absence of the substrate. We suggest that the signal peptide holds/stabilizes the active state conformation of Sec61 during post-translational translocation. Thus, our study explains the effect of Sec62 on the conformation of the Sec61 channel and describes the conformational transitions of Sec61 channel.

How does Sec63 affect the conformation of Sec61 in yeast?

The Sec complex catalyzes the translocation of proteins of the secretory pathway into the endoplasmic reticulum and the integration of membrane proteins into the endoplasmic reticulum membrane. Some substrate peptides require the presence and involvement of accessory proteins such as Sec63. Recently, a structure of the Sec complex from Saccharomyces cerevisiae, consisting of the Sec61 channel and the Sec62, Sec63, Sec71 and Sec72 proteins was determined by cryo-electron microscopy (cryo-EM). Here, we show by co-precipitation that the Sec61 channel subunit Sbh1 is not required for formation of stable Sec63-Sec61 contacts. Molecular dynamics simulations started from the cryo-EM conformation of Sec61 bound to Sec63 and of unbound Sec61 revealed how Sec63 affects the conformation of Sec61 lateral gate, plug, pore region and pore ring diameter via three intermolecular contact regions. Molecular docking of SRP-dependent vs. SRP-independent signal peptide chains into the Sec61 channel showed that the pore regions affected by presence/absence of Sec63 play a crucial role in positioning the signal anchors of SRP-dependent substrates nearby the lateral gate.

Membrane protein biogenesis by the EMC.

The endoplasmic reticulum (ER) membrane protein complex (EMC) was identified over a decade ago in a genetic screen for ER protein homeostasis. The EMC inserts transmembrane domains (TMDs) with limited hydrophobicity. Two recent cryo-EM structures, and a third model based on partial high- and low-resolution structures, suggest how this is accomplished.

The N-terminus of Sec61p plays key roles in ER protein import and ERAD.

Sec61p is the channel-forming subunit of the heterotrimeric Sec61 complex that mediates co-translational protein import into the endoplasmic reticulum (ER). In yeast, proteins can also be post-translationally translocated by the hetero-heptameric Sec complex, composed of the Sec61 and the Sec63 complexes. The Sec61 channel is also a candidate for the dislocation channel for misfolded proteins from the ER to the cytosol during ER-associated degradation (ERAD). The structure of the Sec61 complex is highly conserved, but the roles of its N-terminal acetylation and its amphipathic N-terminal helix are unknown so far. To gain insight into the function of the Sec61p N-terminus, we mutated its N-acetylation site, deleted its amphipathic helix, or both the helix and the N-acetylation site. Mutation of the N-acetylation site on its own had no effect on protein import into the ER in intact cells, but resulted in an ERAD defect. Yeast expressing sec61 without the N-terminal amphipathic helix displayed severe growth defects and had profound defects in post-translational protein import into the ER. Nevertheless the formation of the hetero-heptameric Sec complex was not affected. Instead, the lack of the N-terminal amphipathic helix compromised the integrity of the heterotrimeric Sec61 complex. We conclude that the N-terminal helix of Sec61p is required for post-translational protein import into the ER and Sec61 complex stability, whereas N-terminal acetylation of Sec61p plays a role in ERAD.

Post-Translational Protein Transport by the Sec Complex.

Although it has been studied for 30 years, the mechanism by which secretory proteins are transported post-translationally into the endoplasmic reticulum (ER) has not yet been fully resolved. Recently published structures (Itskanov and Park, Science 2019;363:84-87; Wu, X. et al. Nature 2019;566:136-139) of the heptameric secretory (Sec) complex which mediates post-translational import into the yeast ER shed new light on the process.

Effect of Sec61 interaction with Mpd1 on endoplasmic reticulum-associated degradation.

Proteins that misfold in the endoplasmic reticulum (ER) are transported back to the cytosol for ER-associated degradation (ERAD). The Sec61 channel is one of the candidates for the retrograde transport conduit. Channel opening from the ER lumen must be triggered by ERAD factors and substrates. Here we aimed to identify new lumenal interaction partners of the Sec61 channel by chemical crosslinking and mass spectrometry. In addition to known Sec61 interactors we detected ERAD factors including Cue1, Ubc6, Ubc7, Asi3, and Mpd1. We show that the CPY* ERAD factor Mpd1 binds to the lumenal Sec61 hinge region. Deletion of the Mpd1 binding site reduced the interaction between both proteins and caused an ERAD defect specific for CPY* without affecting protein import into the ER or ERAD of other substrates. Our data suggest that Mpd1 binding to Sec61 is a prerequisite for CPY* ERAD and confirm a role of Sec61 in ERAD of misfolded secretory proteins.

A Case for Sec61 Channel Involvement in ERAD.

Proteins that misfold in the endoplasmic reticulum (ER) need to be transported back to the cytosol for degradation by proteasomes, a process known as ER-associated degradation (ERAD). The first candidate discussed as a retrograde protein transport conduit was the Sec61 channel which is responsible for secretory protein transport into the ER during biogenesis. The Sec61 channel binds the proteasome 19S regulatory particle which can extract an ERAD substrate from the ER. Nevertheless its role as a general export channel has been dismissed, and Hrd1 and Der1 have been proposed as alternatives. The discovery of export-specific sec61 mutants and of mammalian ERAD substrates whose export is dependent on the 19S regulatory particle suggest that dismissal of a role of Sec61 in export may have been premature.

Inhibitors of Protein Translocation Across the ER Membrane.

Protein translocation into the endoplasmic reticulum (ER) constitutes the first step of protein secretion. ER protein import is essential in all eukaryotic cells and is particularly critical in fast-growing tumour cells. Thus, the process can serve as target both for potential cancer drugs and for bacterial virulence factors. Inhibitors of protein transport across the ER membrane range from broad-spectrum to highly substrate-specific and can interfere with virtually any stage of this multistep process, and even with transport of endocytosed antigens into the cytosol for cross-presentation.

Proteasome 19S RP binding to the Sec61 channel plays a key role in ERAD.

Import of secretory proteins into the Endoplasmic Reticulum (ER) is an established function of the Sec61 channel. The contribution of the Sec61 channel to export of misfolded proteins from the ER for degradation by proteasomes is still controversial, but the proteasome 19S regulatory particle (RP) is necessary and sufficient for extraction of specific misfolded proteins from the ER, and binds directly to the Sec61 channel. In this work we have identified an import-competent sec61 mutant, S353C, carrying a point mutation in ER-lumenal loop 7 which reduces affinity of the cytoplasmic face of the Sec61 channel for the 19S RP. This indicates that the interaction between the 19S RP and the Sec61 channel is dependent on conformational changes in Sec61p hinging on loop 7. The sec61-S353C mutant had no measurable ER import defects and did not cause ER stress in intact cells, but reduced ER-export of a 19S RP-dependent misfolded protein when proteasomes were limiting in a cell-free assay. Our data suggest that the interaction between the 19S RP and the Sec61 channel is essential for the export of specific substrates from the ER to the cytosol for proteasomal degradation.

ERAD and protein import defects in a sec61 mutant lacking ER-lumenal loop 7.

BACKGROUND: The Sec61 channel mediates protein translocation across the endoplasmic reticulum (ER) membrane during secretory protein biogenesis, and likely also during export of misfolded proteins for ER-associated degradation (ERAD). The mechanisms of channel opening for the different modes of translocation are not understood so far, but the position of the large ER-lumenal loop 7 of Sec61p suggests a decisive role. RESULTS: We show here that the Y345H mutation in L7 which causes diabetes in the mouse displays no ER import defects in yeast, but a delay in misfolded protein export. A complete deletion of L7 in Sec61p resulted in viable, cold- and tunicamycin-hypersensitive yeast cells with strong defects in posttranslational protein import of soluble proteins into the ER, and in ERAD of soluble substrates. Membrane protein ERAD was only moderately slower in sec61∆L7 than in wildtype cells. Although Sec61∆L7 channels were unstable in detergent, co-translational protein integration into the ER membrane, proteasome binding to Sec61∆L7 channels, and formation of hetero-heptameric Sec complexes were not affected. CONCLUSIONS: We conclude that L7 of Sec61p is required for initiation of posttranslational soluble protein import into and misfolded soluble protein export from the ER, suggesting a key role for L7 in transverse gating of the Sec61 channel.

The Sec63p J-domain is required for ERAD of soluble proteins in yeast.

How misfolded proteins are exported from the ER to the cytosol for degradation (ER-associated Degradation, ERAD) and which proteins are participating in this process is not understood. Several studies using a single, leaky mutant indicated that Sec63p might be involved in ERAD. More recently, Sec63p was also found strongly associated with proteasomes attached to the protein-conducting channel in the ER membrane which presumably form part of the export machinery. These observations prompted us to reinvestigate the role of Sec63p in ERAD by generating new mutants which were selected in a screen monitoring the intracellular accumulation of the ERAD substrate CPY*. We show that a mutation in the DnaJ-domain of Sec63p causes a defect in ERAD, whereas mutations in the Brl, acidic, and transmembrane domains only affect protein import into the ER. Unexpectedly, mutations in the acidic domain which mediates interaction of Sec63p with Sec62p also caused defects in cotranslational import. In contrast to mammalian cells where SEC63 expression levels affect steady-state levels of multi-spanning transmembrane proteins, the sec63 J-domain mutant was only defective in ERAD of soluble substrates.

Analysis of Sec61p and Ssh1p interactions in the ER membrane using the split-ubiquitin system.

BACKGROUND: The split-ubiquitin system monitors interactions of transmembrane proteins in yeast. It is based on the formation of a quasi-native ubiquitin structure upon interaction of two proteins to which the N- and C-terminal halves of ubiquitin have been fused. In the system we use here ubiquitin formation leads to proteolytic cleavage liberating a transcription factor (PLV) from the C-ubiquitin (C) fusion protein which can then activate reporter genes. Generation of fusion proteins is, however, rife with problems, and particularly in transmembrane proteins often disturbs topology, structure and function. RESULTS: We show that both the Sec61 protein which forms the principal protein translocation channel in the endoplasmic reticulum (ER) membrane, and its non-essential homologue, Ssh1p, when fused C-terminally to CPLV are inactive. In a heterozygous diploid Sec61-CPLV is present in protein translocation channels in the ER membrane without disturbing their function and displays a limited set of protein-protein interactions similar to those found for the wildtype protein using biochemical methods. Although its expression level is similar, Ssh1-CPLV interactions are less strong, and, in contrast to Sec61p, Ssh1p does not distinguish between Sbh1p and Sbh2p. We show that interactions can be monitored by reporter gene activity or directly by PLV cleavage, which is more sensitive, but leads to quantitatively different results. CONCLUSIONS: We conclude that the split-ubiquitin system we used here has high fidelity, but low sensitivity and is of limited use for detection of new, transient interactions with protein translocation channels in the ER membrane.

N-acetylation and phosphorylation of Sec complex subunits in the ER membrane.

BACKGROUND: Covalent modifications of proteins provide a mechanism to control protein function. Here, we have investigated modifications of the heptameric Sec complex which is responsible for post-translational protein import into the endoplasmic reticulum (ER). It consists of the Sec61 complex (Sec61p, Sbh1p, Sss1p) which on its own mediates cotranslational protein import into the ER and the Sec63 complex (Sec63p, Sec62p, Sec71p, Sec72p). Little is known about the biogenesis and regulation of individual Sec complex subunits. RESULTS: We show that Sbh1p when it is part of the Sec61 complex is phosphorylated on T5 which is flanked by proline residues. The phosphorylation site is conserved in mammalian Sec61ß, but only partially in birds, and not in other vertebrates or unicellular eukaryotes, suggesting convergent evolution. Mutation of T5 to A did not affect the ability of mutant Sbh1p to complement the growth defect in a Δsbh1Δsbh2 strain, and did not result in a hypophosphorylated protein which shows that alternate sites can be used by the T5 kinase. A survey of yeast phosphoproteome data shows that Sbh1p can be phosphorylated on multiple sites which are organized in two patches, one at the N-terminus of its cytosolic domain, the other proximal to the transmembrane domain. Surprisingly, although N-acetylation has been shown to interfere with ER targeting, we found that both Sbh1p and Sec62p are cotranslationally N-acetylated by NatA, and N-acetyl-proteome data indicate that Sec61p is modified by the same enzyme. Mutation of the N-acetylation site, however, did not affect Sec62p function in posttranslational protein import into the ER. Disabling NatA resulted in growth retardation, but not in co- or posttranslational translocation defects or instability of Sec62p or Sbh1p. CONCLUSIONS: We conclude that N-acetylation of transmembrane and tail-anchored proteins does not interfere with their ER-targeting, and that Sbh1p phosphorylation on T5, which is not present in Sbh2p, plays a non-essential role specific to the Sec61 complex.

Diversion at the ER: How Plasmodium falciparum exports proteins into host erythrocytes.

Malaria is caused by parasites which live in host erythrocytes and remodel these cells to provide optimally for the parasites' needs by exporting effector proteins into the host cells. Eight years ago the discovery of a host cell targeting sequence present in both soluble and transmembrane  P. falciparum exported proteins generated a starting point for investigating the mechanism of parasite protein transport into infected erythrocytes. Since then many confusing facts about this targeting signal have emerged. In this paper, I try to make sense of them.

The intracellular accumulation of polymeric neuroserpin explains the severity of the dementia FENIB.

Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is an autosomal dominant dementia that is characterized by the retention of polymers of neuroserpin as inclusions within the endoplasmic reticulum (ER) of neurons. We have developed monoclonal antibodies that detect polymerized neuroserpin and have used COS-7 cells, stably transfected PC12 cell lines and transgenic Drosophila melanogaster to characterize the cellular handling of all four mutant forms of neuroserpin that cause FENIB. We show a direct correlation between the severity of the disease-causing mutation and the accumulation of neuroserpin polymers in cell and fly models of the disease. Moreover, mutant neuroserpin causes locomotor deficits in the fly allowing us to demonstrate a direct link between polymer accumulation and neuronal toxicity.

The transmembrane domain is sufficient for Sbh1p function, its association with the Sec61 complex, and interaction with Rtn1p.

The Sec61 protein translocation complex in the endoplasmic reticulum (ER) membrane is composed of three subunits. The alpha-subunit, called Sec61p in yeast, is a multispanning membrane protein that forms the protein conducting channel. The functions of the smaller, carboxyl-terminally tail-anchored beta subunit Sbh1p, its close homologue Sbh2p, and the gamma subunit Sss1p are not well understood. Here we show that co-translational protein translocation into the ER is reduced in sbh1Delta sbh2Delta cells, whereas there is a limited reduction of post-translational translocation and no effect on export of a mutant form of alpha-factor precursor for ER-associated degradation in the cytosol. The translocation defect and the temperature-sensitive growth phenotype of sbh1Delta sbh2Delta cells were rescued by expression of the transmembrane domain of Sbh1p alone, and the Sbh1p transmembrane domain was sufficient for coimmunoprecipitation with Sec61p and Sss1p. Furthermore, we show that Sbh1p co-precipitates with the ER transmembrane protein Rtn1p. Sbh1p-Rtn1p complexes do not appear to contain Sss1p and Sec61p. Our results define the transmembrane domain as the minimal functional domain of the Sec61beta homologue Sbh1p in ER translocation, identify a novel interaction partner for Shb1p, and imply that Sbh1p has additional functions that are not directly linked to protein translocation in association with the Sec61 complex.

Characterization of the proteasome interaction with the Sec61 channel in the endoplasmic reticulum.

Biogenesis of secretory proteins requires their translocation into the endoplasmic reticulum (ER) through the Sec61 channel. Proteins that fail to fold are transported back into the cytosol and are degraded by proteasomes. For many substrates this retrograde transport is affected by mutations in the Sec61 channel, and can be promoted by ATP and the 19S regulatory particle of the proteasome, which binds directly to the Sec61 channel via its base. Here, we identify mutations in SEC61 which reduce proteasome binding to the channel, and demonstrate that proteasomes and ribosomes bind differently to cytosolic domains of the channel. We found that Sec63p and BiP coprecipitate with ER-associated proteasomes, but Sec63p does not contribute to proteasome binding to the ER. The 19S base contains six AAA-ATPase subunits (Rpt proteins) that have non-equivalent functions in proteasome-mediated protein turnover and form a hetero-hexamer. Mutations in the ATP-binding sites of individual Rpt proteins all reduced the affinity of 19S complexes for the ER, suggesting that the 19S base in the ATP-bound conformation docks at the Sec61 channel.

Human autoantibodies against the 54 kDa protein of the signal recognition particle block function at multiple stages.

The 54 kDa subunit of the signal recognition particle (SRP54) binds to the signal sequences of nascent secretory and membrane proteins and it contributes to the targeting of these precursors to the membrane of the endoplasmic reticulum (ER). At the ER membrane, the binding of the signal recognition particle (SRP) to its receptor triggers the release of SRP54 from its bound signal sequence and the nascent polypeptide is transferred to the Sec61 translocon for insertion into, or translocation across, the ER membrane. In the current article, we have characterized the specificity of anti-SRP54 autoantibodies, which are highly characteristic of polymyositis patients, and investigated the effect of these autoantibodies on the SRP function in vitro. We found that the anti-SRP54 autoantibodies had a pronounced and specific inhibitory effect upon the translocation of the secretory protein preprolactin when analysed using a cell-free system. Our mapping studies showed that the anti-SRP54 autoantibodies bind to the amino-terminal SRP54 N-domain and to the central SRP54 G-domain, but do not bind to the carboxy-terminal M-domain that is known to bind ER signal sequences. Nevertheless, anti-SRP54 autoantibodies interfere with signal-sequence binding to SRP54, most probably by steric hindrance. When the effect of anti-SRP autoantibodies on protein targeting the ER membrane was further investigated, we found that the autoantibodies prevent the SRP receptor-mediated release of ER signal sequences from the SRP54 subunit. This observation supports a model where the binding of the homologous GTPase domains of SRP54 and the alpha-subunit of the SRP receptor to each other regulates the release of ER signal sequences from the SRP54 M-domain.