Translocon-associated Protein Research Articles

Role of Sec61α2 Translocon in Insulin Biosynthesis.

Translocational regulation of proinsulin biosynthesis in pancreatic β-cells is unknown, although several studies have reported an important accessory role for the Translocon-Associated Protein complex to assist preproinsulin delivery into the endoplasmic reticulum via the heterotrimeric Sec61 translocon (comprised of α, β, and γ subunits). The actual protein-conducting channel is the α-subunit encoded either by Sec61A1 or its paralog Sec61A2. Although the underlying channel selectivity for preproinsulin translocation is unknown, almost all studies of Sec61α to date have focused on Sec61α1. There is currently no evidence to suggest that this gene product plays a major role in proinsulin production, whereas genome-wide association studies indicate linkage of Sec61A2 with diabetes. Here, we report that evolutionary differences in mouse preproinsulin signal peptides affect proinsulin biosynthesis. Moreover, we find that although some preproinsulin translocation can proceed through Sec61α1, Sec61α2 has a greater impact on proinsulin biosynthesis in pancreatic β-cells. Remarkably, Sec61α2-translocon deficiency exerts a significant inhibitory effect on the biosynthesis of preproinsulin itself, including a disproportionate increase of full-length nacent chain unreleased from ribosomes. This study not only reveals novel translocational regulation of proinsulin biosynthesis, but also provides a rationale for genetic evidence suggesting an important role of Sec61α2 in maintaining blood glucose homeostasis.

Structural analysis of the dynamic ribosome-translocon complex

The protein translocon at the endoplasmic reticulum comprises the Sec61 translocation channel and numerous accessory factors that collectively facilitate the biogenesis of secretory and membrane proteins. Here, we leveraged recent advances in cryo-electron microscopy (cryo-EM) and structure prediction to derive insights into several novel configurations of the ribosome-translocon complex. We show how a transmembrane domain (TMD) in a looped configuration passes through the Sec61 lateral gate during membrane insertion; how a nascent chain can bind and constrain the conformation of ribosomal protein uL22; and how the translocon-associated protein (TRAP) complex can adjust its position during different stages of protein biogenesis. Most unexpectedly, we find that a large proportion of translocon complexes contains RAMP4 intercalated into Sec61’s lateral gate, widening Sec61’s central pore and contributing to its hydrophilic interior. These structures lead to mechanistic hypotheses for translocon function and highlight a remarkably plastic machinery whose conformations and composition adjust dynamically to its diverse range of substrates.

Structural analysis of the dynamic ribosome-translocon complex.

The protein translocon at the endoplasmic reticulum comprises the Sec61 translocation channel and numerous accessory factors that collectively facilitate the biogenesis of secretory and membrane proteins. Here, we leveraged recent advances in cryo-electron microscopy (cryo-EM) and structure prediction to derive insights into several novel configurations of the ribosome-translocon complex. We show how a transmembrane domain (TMD) in a looped configuration passes through the Sec61 lateral gate during membrane insertion; how a nascent chain can bind and constrain the conformation of ribosomal protein uL22; and how the translocon-associated protein (TRAP) complex can adjust its position during different stages of protein biogenesis. Most unexpectedly, we find that a large proportion of translocon complexes contains RAMP4 intercalated into Sec61's lateral gate, widening Sec61's central pore and contributing to its hydrophilic interior. These structures lead to mechanistic hypotheses for translocon function and highlight a remarkably plastic machinery whose conformations and composition adjust dynamically to its diverse range of substrates.

Exploring the molecular composition of the multipass translocon in its native membrane environment.

Multispanning membrane proteins are inserted into the endoplasmic reticulum membrane by the ribosome-bound multipass translocon (MPT) machinery. Based on cryo-electron tomography and extensive subtomogram analysis, we reveal the composition and arrangement of ribosome-bound MPT components in their native membrane environment. The intramembrane chaperone complex PAT and the translocon-associated protein (TRAP) complex associate substoichiometrically with the MPT in a translation-dependent manner. Although PAT is preferentially part of MPTs bound to translating ribosomes, the abundance of TRAP is highest in MPTs associated with non-translating ribosomes. The subtomogram average of the TRAP-containing MPT reveals intermolecular contacts between the luminal domains of TRAP and an unknown subunit of the back-of-Sec61 complex. AlphaFold modeling suggests this protein is nodal modulator, bridging the luminal domains of nicalin and TRAPα. Collectively, our results visualize the variability of MPT factors in the native membrane environment dependent on the translational activity of the bound ribosome.

Molecular view of ER membrane remodeling by the Sec61/TRAP translocon.

Protein translocation across the endoplasmic reticulum (ER) membrane is an essential step during protein entry into the secretory pathway. The conserved Sec61 protein-conducting channel facilitates polypeptide translocation and coordinates cotranslational polypeptide-processing events. In cells, the majority of Sec61 is stably associated with a heterotetrameric membrane protein complex, the translocon-associated protein complex (TRAP), yet the mechanism by which TRAP assists in polypeptide translocation remains unknown. Here, we present the structure of the core Sec61/TRAP complex bound to a mammalian ribosome by cryogenic electron microscopy (cryo-EM). Ribosome interactions anchor the Sec61/TRAP complex in a conformation that renders the ER membrane locally thinner by significantly curving its lumenal leaflet. We propose that TRAP stabilizes the ribosome exit tunnel to assist nascent polypeptide insertion through Sec61 and provides a ratcheting mechanism into the ER lumen mediated by direct polypeptide interactions.

1752-P: The Role of Translocon-Associated Protein Subunit γ in Insulin Biosynthesis and Glucose Homeostasis

Translocation-associated protein (TRAP) is a four-subunit complex located on the endoplasmic reticulum (ER) membrane, including TRAPα, TRAPβ, TRAPγ, and TRAPδ. TRAPα, TRAPβ and TRAPδ reside on the luminal side of the ER membrane for primary functionality, whereas the extra-membranous portion of TRAPγ is mainly cytosolic. Our recent studies indicate that TRAP complex contributes to the biosynthesis of proinsulin in pancreatic β cell lines and TRAPγ is a rate-limiting element of proinsulin biosynthesis. This might be partially explained by that newly made preproinsulin may first encounter the cytosolic domain of TRAPγ for translocation into the ER. To further understand the role of TRAPγ in β cells and its pathological significance in maintaining glucose homeostasis, we generated a β-cell specific TRAPγ knockout mouse model. We found that TRAPγ deficiency led to a significant reduction of insulin content in islets and a decrease of circulating insulin. TRAPγ βKO mice impaired glucose tolerance with aging, which even further deteriorated with high fat diet. In β cell line, TRAPγ deficiency resulted in less proinsulin synthesis and poor response to high glucose stimulation. Consistently, an acute exposure to high glucose increased expression of TRAPγ and proinsulin protein levels simultaneously in wild-type mouse islets. However, the ability to upregulate proinsulin response to high glucose was markedly compromised in TRAPγ βKO mouse islets. This study uncovers that TRAPγ is critical to insulin synthesis both in vivo and in vitro, which provides insight into the pathogenesis of type 2 Diabetes. Disclosure N.Xu: None. X.Xu: None. J.Hu: None. J.Guo: None. J.Cui: None. M.Liu: None. W.Feng: None. X.Li: None. Funding National Natural Science Foundation of China (81830025, 82220108014, 82100865, 81800733, 81900720, 82000796, 82070854); National Key R&D Program of China (2019YFA0802502, 2022YFE0131400); Tianjin Key Medical Discipline Construction Project (TJYXZDXK-030A)

Molecular basis of the TRAP complex function in ER protein biogenesis

The translocon-associated protein (TRAP) complex resides in the endoplasmic reticulum (ER) membrane and interacts with the Sec translocon and the ribosome to facilitate biogenesis of secretory and membrane proteins. TRAP plays a key role in the secretion of many hormones, including insulin. Here we reveal the molecular architecture of the mammalian TRAP complex and how it engages the translating ribosome associated with Sec61 translocon on the ER membrane. The TRAP complex is anchored to the ribosome via a long tether and its position is further stabilized by a finger-like loop. This positions a cradle-like lumenal domain of TRAP below the translocon for interactions with translocated nascent chains. Our structure-guided TRAP mutations in Caenorhabditis elegans lead to growth deficits associated with increased ER stress and defects in protein hormone secretion. These findings elucidate the molecular basis of the TRAP complex in the biogenesis and translocation of proteins at the ER.

Structural insights into TRAP association with ribosome-Sec61 complex and translocon inhibition by a CADA derivative.

During cotranslational translocation, the signal peptide of a nascent chain binds Sec61 translocon to initiate protein transport through the endoplasmic reticulum (ER) membrane. Our cryo-electron microscopy structure of ribosome-Sec61 shows binding of an ordered heterotetrameric translocon-associated protein (TRAP) complex, in which TRAP-γ is anchored at two adjacent positions of 28S ribosomal RNA and interacts with ribosomal protein L38 and Sec61α/γ. Four transmembrane helices (TMHs) of TRAP-γ cluster with one C-terminal helix of each α, β, and δ subunits. The seven TMH bundle helps position a crescent-shaped trimeric TRAP-α/β/δ core in the ER lumen, facing the Sec61 channel. Further, our in vitro assay establishes the cyclotriazadisulfonamide derivative CK147 as a translocon inhibitor. A structure of ribosome-Sec61-CK147 reveals CK147 binding the channel and interacting with the plug helix from the lumenal side. The CK147 resistance mutations surround the inhibitor. These structures help in understanding the TRAP functions and provide a new Sec61 site for designing translocon inhibitors.

Visualization of translation and protein biogenesis at the ER membrane

The dynamic ribosome–translocon complex, which resides at the endoplasmic reticulum (ER) membrane, produces a major fraction of the human proteome1,2. It governs the synthesis, translocation, membrane insertion, N-glycosylation, folding and disulfide-bond formation of nascent proteins. Although individual components of this machinery have been studied at high resolution in isolation3–7, insights into their interplay in the native membrane remain limited. Here we use cryo-electron tomography, extensive classification and molecular modelling to capture snapshots of mRNA translation and protein maturation at the ER membrane at molecular resolution. We identify a highly abundant classical pre-translocation intermediate with eukaryotic elongation factor 1a (eEF1a) in an extended conformation, suggesting that eEF1a may remain associated with the ribosome after GTP hydrolysis during proofreading. At the ER membrane, distinct polysomes bind to different ER translocons specialized in the synthesis of proteins with signal peptides or multipass transmembrane proteins with the translocon-associated protein complex (TRAP) present in both. The near-complete atomic model of the most abundant ER translocon variant comprising the protein-conducting channel SEC61, TRAP and the oligosaccharyltransferase complex A (OSTA) reveals specific interactions of TRAP with other translocon components. We observe stoichiometric and sub-stoichiometric cofactors associated with OSTA, which are likely to include protein isomerases. In sum, we visualize ER-bound polysomes with their coordinated downstream machinery.

Methionine Restriction Impairs Degradation of a Protein that Aberrantly Engages the Endoplasmic Reticulum Translocon.

Proteins that persistently engage endoplasmic reticulum (ER) translocons are degraded by multiple translocon quality control (TQC) mechanisms. In Saccharomyces cerevisiae , the model translocon-associated protein Deg1 -Sec62 is subject to ER-associated degradation (ERAD) by the Hrd1 ubiquitin ligase and, to a lesser extent, proteolysis mediated by the Ste24 protease. In a recent screen, we identified nine methionine-biosynthetic genes as candidate TQC regulators. Here, we found methionine restriction impairs Hrd1-independent Deg1 -Sec62 degradation. Beyond revealing methionine as a novel regulator of TQC, our results urge caution when working with laboratory yeast strains with auxotrophic mutations, often presumed not to influence cellular processes under investigation.

Translocon-associated Protein Subunit SSR3 Determines and Predicts Susceptibility to Paclitaxel in Breast Cancer and Glioblastoma.

Paclitaxel (PTX) is one of the most potent and commonly used chemotherapies for breast and pancreatic cancer. Several ongoing clinical trials are investigating means of enhancing delivery of PTX across the blood-brain barrier for glioblastomas. Despite the widespread use of PTX for breast cancer, and the initiative to repurpose this drug for gliomas, there are no predictive biomarkers to inform which patients will likely benefit from this therapy. To identify predictive biomarkers for susceptibility to PTX, we performed a genome-wide CRISPR knockout (KO) screen using human glioma cells. The genes whose KO was most enriched in the CRISPR screen underwent further selection based on their correlation with survival in the breast cancer patient cohorts treated with PTX and not in patients treated with other chemotherapies, a finding that was validated on a second independent patient cohort using progression-free survival. Combination of CRISPR screen results with outcomes from patients with taxane-treated breast cancer led to the discovery of endoplasmic reticulum (ER) protein SSR3 as a putative predictive biomarker for PTX. SSR3 protein levels showed positive correlation with susceptibility to PTX in breast cancer cells, glioma cells, and in multiple intracranial glioma xenografts models. KO of SSR3 turned the cells resistant to PTX while its overexpression sensitized the cells to PTX. Mechanistically, SSR3 confers susceptibility to PTX through regulation of phosphorylation of ER stress sensor IRE1α. Our hypothesis generating study showed SSR3 as a putative biomarker for susceptibility to PTX, warranting its prospective clinical validation.

SSR4 as a prognostic biomarker and related with immune infiltration cells in colon adenocarcinoma

Background Signal sequence receptor subunit delta (SSR4) gene is reported to encode the translocon-associated protein δ, and related with the human immune regulation. However, the expression of SSR4 in colon adenocarcinoma (COAD) and its correlation with clinical treatment remains unclear. Research design and methods SSR4 mRNA expression level and its relationship with tumor infiltrating lymphocytes (TILs) in COAD were evaluated through several databases. Furthermore, the study collected 238 cases of COAD tissue samples to detect the association of SSR4 protein expression level in TILs with clinical pathological information, and the prognosis of COAD. Results SSR4 mRNA was significantly high expressed in COAD tissues, and significantly correlated with several types of TILs in COAD. Moreover, SSR4 highly expressed in many types of TILs, especially highly expressed in plasma cell from COAD patients with advanced TNM stage. High SSR4 protein expression in TILs was associated with lymph node metastasis, distant metastasis, American Joint Committee on Cancer (AJCC) staging, and Response Evaluation Criteria in Solid Tumors (RECIST) efficacy. COAD patients with high SSR4 expression in TILs had better overall survival. Conclusions In conclusion, high SSR4 mRNA and protein expression in TILs can be used as a prognostic biomarker for predicting better overall survival and treatment efficacy in COAD patients.

Biogenesis of hepatitis B virus e antigen is driven by translocon-associated protein complex and regulated by conserved cysteine residues within its signal peptide sequence.

Hepatitis B virus uses e antigen (HBe), which is dispensable for virus infectivity, to modulate host immune responses and achieve viral persistence in human hepatocytes. The HBe precursor (p25) is directed to the endoplasmic reticulum (ER), where cleavage of the signal peptide (sp) gives rise to the first processing product, p22. P22 can be retro‐translocated back to the cytosol or enter the secretory pathway and undergo a second cleavage event, resulting in secreted p17 (HBe). Here, we report that translocation of p25 to the ER is promoted by translocon‐associated protein complex. We have found that p25 is not completely translocated into the ER; a fraction of p25 is phosphorylated and remains in the cytoplasm and nucleus. Within the p25 sp sequence, we have identified three cysteine residues that control the efficiency of sp cleavage and contribute to proper subcellular distribution of the precore pool.

Deficient endoplasmic reticulum translocon-associated protein complex limits the biosynthesis of proinsulin and insulin.

The conserved endoplasmic reticulum (ER) membrane protein TRAPα (translocon-associated protein, also known as signal sequence receptor 1, SSR1) has been reported to play a critical but unclear role in insulin biosynthesis. TRAPα/SSR1 is one component of a four-protein complex including TRAPβ/SSR2, TRAPγ/SSR3, and TRAPδ/SSR4. The TRAP complex topologically has a small exposure on the cytosolic side of the ER via its TRAPγ/SSR3 subunit, whereas TRAPβ/SSR2 and TRAPδ/SSR4 function along with TRAPα/SSR1 largely on the luminal side of the ER membrane. Here, we have examined pancreatic β-cells with deficient expression of either TRAPβ/SSR2 or TRAPδ/SSR4, which does not perturb mRNA expression levels of other TRAP subunits, or insulin mRNA. However, deficient protein expression of TRAPβ/SSR2 and, to a lesser degree, TRAPδ/SSR4, diminishes the protein levels of other TRAP subunits, concomitant with deficient steady-state levels of proinsulin and insulin. Deficient TRAPβ/SSR2 or TRAPδ/SSR4 is not associated with any apparent defect of exocytotic mechanism but rather by a decreased abundance of the proinsulin and insulin that accompanies glucose-stimulated secretion. Amino acid pulse labeling directly establishes that much of the steady-state deficiency of intracellular proinsulin can be accounted for by diminished proinsulin biosynthesis, observed in a pulse-labeling as short as 5minutes. The proinsulin and insulin levels in TRAPβ/SSR2 or TRAPδ/SSR4 null mutant β-cells are notably recovered upon re-expression of the missing TRAP subunit, accompanying a rebound of proinsulin biosynthesis. Remarkably, overexpression of TRAPα/SSR1 can also suppress defects in β-cells with diminished expression of TRAPβ/SSR2, strongly suggesting that TRAPβ/SSR2 is needed to support TRAPα/SSR1 function.

The translocon-associated protein (TRAP) complex regulates quality control of N-linked glycosylation during ER stress.

Asparagine (N)-linked glycosylation is required for endoplasmic reticulum (ER) homeostasis, but how this co- and posttranslational modification is maintained during ER stress is unknown. Here, we introduce a fluorescence-based strategy to detect aberrant N-glycosylation in individual cells and identify a regulatory role for the heterotetrameric translocon-associated protein (TRAP) complex. Unexpectedly, cells with knockout of SSR3 or SSR4 subunits restore N-glycosylation over time concurrent with a diminished ER stress transcriptional signature. Activation of ER stress or silencing of the ER chaperone BiP exacerbates or rescues the glycosylation defects, respectively, indicating that SSR3 and SSR4 enable N-glycosylation during ER stress. Protein levels of the SSR3 subunit are ER stress and UBE2J1 dependent, revealing a mechanism that coordinates upstream N-glycosylation proficiency with downstream ER-associated degradation and proteostasis. The fidelity of N-glycosylation is not static in both nontransformed and tumor cells, and the TRAP complex regulates ER glycoprotein quality control under conditions of stress.

Expanding the phenotype of X-linked SSR4-CDG: Connective tissue implications.

Signal sequence receptor protein 4 (SSR4) is a subunit of the translocon-associated protein complex, which participates in the translocation of proteins across the endoplasmic reticulum membrane, enhancing the efficiency of N-linked glycosylation. Pathogenic variants in SSR4 cause a congenital disorder of glycosylation: SSR4-congenital disorders of glycosylation (CDG). We describe three SSR4-CDG boys and review the previously reported. All subjects presented with hypotonia, failure to thrive, developmental delay, and dysmorphic traits and showed a type 1 serum sialotransferrin profile, facilitating the diagnosis. Genetic confirmation of this X-linked CDG revealed one de novo hemizygous deletion, one maternally inherited deletion, and one de novo nonsense mutation of SSR4. The present subjects highlight the similarities with a connective tissue disorder (redundant skin, joint laxity, blue sclerae, and vascular tortuosity). The connective tissue problems are relevant, and require preventive rehabilitation measures. As an X-linked disorder, genetic counseling is essential.

Translocon-Associated Protein Complex (TRAP) is Crucial for Co-Translational Translocation of Pre-Proinsulin

Many unanswered questions remain in understanding the biosynthesis of the peptide hormone insulin. Here we elucidate new aspects in the mechanism of co-translational translocation initiation of pre-proinsulin in the endoplasmic reticulum. We utilize a translational arrest peptide derived from the x-box-binding protein (Xbp1) to induce ribosomal stalling and generate translocation intermediates. We find that the insulin signal sequence is rather weakly gating and requires the assistance of auxiliary translocon components to initiate translocation. Probing the translational intermediates with chemical crosslinking, we identified an early interaction with the translocon-associated protein (TRAP) complex. The TRAPβ subunit interacts with pre-proinsulin before the peptide enters the Sec61 translocon channel in a signal sequence-dependent manner. We describe the substrate sequence determinants that are recognized by TRAP on the cytosolic site of the membrane to facilitate substrate-specific opening of the Sec61 translocon channel. Our findings support the hypothesis that the TRAP-dependence is in part determined by the content of glycine and proline residues mainly within the signal sequence.

Understanding the mammalian TRAP complex function(s).

In eukaryotic cells, about one-third of the synthesized proteins are translocated into the endoplasmic reticulum; they are membrane or lumen resident proteins and proteins direct to the Golgi apparatus. The co-translational translocation takes place through the heterotrimeric protein-conducting channel Sec61 which is associated with the ribosome and many accessory components, such as the heterotetrameric translocon-associated protein (TRAP) complex. Recently, microscopic techniques, such as cryo-electron microscopy and cryo-electron tomography, have enabled the determination of the translocation machinery structure. However, at present, there is a lack of understanding regarding the roles of some of its components; indeed, the TRAP complex function during co-translational translocation needs to be established. In addition, TRAP may play a role during unfolded protein response, endoplasmic-reticulum-associated protein degradation and congenital disorder of glycosylation (ssr4 CDG). In this article, I describe the current understanding of the TRAP complex in the light of its possible function(s).

Endoplasmic reticulum stress-regulated degradation of a translocon-associated protein is independent of integrated stress response transcription factor Gcn4p.

Endoplasmic reticulum stress-regulated degradation of a translocon-associated protein is independent of integrated stress response transcription factor Gcn4p.

Protein Translocation Acquires Substrate Selectivity Through ER Stress-Induced Reassembly of Translocon Auxiliary Components.

Protein import across the endoplasmic reticulum membrane is physiologically regulated in a substrate-selective manner to ensure the protection of stressed ER from the overload of misfolded proteins. However, it is poorly understood how different types of substrates are accurately distinguished and disqualified during translocational regulation. In this study, we found poorly assembled translocon-associated protein (TRAP) complexes in stressed ER. Immunoaffinity purification identified calnexin in the TRAP complex in which poor assembly inhibited membrane insertion of the prion protein (PrP) in a transmembrane sequence-selective manner, through translocational regulation. This reaction was induced selectively by redox perturbation, rather than calcium depletion, in the ER. The liberation of ERp57 from calnexin appeared to be the reason for the redox sensitivity. Stress-independent disruption of the TRAP complex prevented a pathogenic transmembrane form of PrP (ctmPrP) from accumulating in the ER. This study uncovered a previously unappreciated role for calnexin in assisting the redox-sensitive function of the TRAP complex and provided insights into the ER stress-induced reassembly of translocon auxiliary components as a key mechanism by which protein translocation acquires substrate selectivity.