Translocon-associated Protein Complex Research Articles

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.

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.

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.

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.

A clearer picture of the ER translocon complex.

The endoplasmic reticulum (ER) translocon complex is the main gate into the secretory pathway, facilitating the translocation of nascent peptides into the ER lumen or their integration into the lipid membrane. Protein biogenesis in the ER involves additional processes, many of them occurring co-translationally while the nascent protein resides at the translocon complex, including recruitment of ER-targeted ribosome-nascent-chain complexes, glycosylation, signal peptide cleavage, membrane protein topogenesis and folding. To perform such varied functions on a broad range of substrates, the ER translocon complex has different accessory components that associate with it either stably or transiently. Here, we review recent structural and functional insights into this dynamically constituted central hub in the ER and its components. Recent cryo-electron microscopy (EM) studies have dissected the molecular organization of the co-translational ER translocon complex, comprising the Sec61 protein-conducting channel, the translocon-associated protein complex and the oligosaccharyl transferase complex. Complemented by structural characterization of the post-translational import machinery, key molecular principles emerge that distinguish co- and post-translational protein import and biogenesis. Further cryo-EM structures promise to expand our mechanistic understanding of the various biochemical functions involving protein biogenesis and quality control in the ER.

Dissecting the molecular organization of the translocon-associated protein complex

In eukaryotic cells, one-third of all proteins must be transported across or inserted into the endoplasmic reticulum (ER) membrane by the ER protein translocon. The translocon-associated protein (TRAP) complex is an integral component of the translocon, assisting the Sec61 protein-conducting channel by regulating signal sequence and transmembrane helix insertion in a substrate-dependent manner. Here we use cryo-electron tomography (CET) to study the structure of the native translocon in evolutionarily divergent organisms and disease-linked TRAP mutant fibroblasts from human patients. The structural differences detected by subtomogram analysis form a basis for dissecting the molecular organization of the TRAP complex. We assign positions to the four TRAP subunits within the complex, providing insights into their individual functions. The revealed molecular architecture of a central translocon component advances our understanding of membrane protein biogenesis and sheds light on the role of TRAP in human congenital disorders of glycosylation.

Single Copies of Sec61 and TRAP Associate with a Nontranslating Mammalian Ribosome

During cotranslational protein translocation, the ribosome associates with a membrane channel, formed by the Sec61 complex, and recruits the translocon-associated protein complex (TRAP). Here we report the structure of a ribosome-channel complex from mammalian endoplasmic reticulum in which the channel has been visualized at 11 A resolution. In this complex, single copies of Sec61 and TRAP associate with a nontranslating ribosome and this stoichiometry was verified by quantitative mass spectrometry. A bilayer-like density surrounds the channel and can be attributed to lipid and detergent. The crystal structure of an archaeal homolog of the Sec61 complex was then docked into the map. In this model, two cytoplasmic loops of Sec61 may interact with RNA helices H6, H7, and H50, while the central pore is located below the ribosome tunnel exit. Hence, this copy of Sec61 is positioned to capture and translocate the nascent chain. Finally, we show that mammalian and bacterial ribosome-channel complexes have similar architectures.

Structure and sequential gene expression of in vitro-induced pronephros (kidney) in the amphibian development

We have successfully induced the formation of 20 different organs in vitro from animal caps (undifferentiated or multipotent cells) of Xenopus blastulae and six different tissues from mice embryonic stem cells. For the Xenopus embryo, we subsequently have attempted to transplant these in vitro-generated organs into embryos to determine their functional capacity. In the experiment we induced the pronephros (nephron) consisting of renal tubule, glomerulus, and renal duct from animal caps treated with activin and retinoic acid. Then we transplanted these cell mass treated with activin and retinoic acid into an embryo in which the renal rudiment had been removed. We found that the transplant developed into pronephros normally and function in vivo. Using this system, we have cloned and analyzed the pronephros-specific genes in Xenopus development. Sequential gene expressions of pronephros in vivo and in vitro resemble the mesonephros formation in mammals. In the screening for genes expressed in pronephroi, we isolated a novel gene, named dullard. Dullard protein contains the C-terminal conserved domain of nuclear LIM interactor-interacting factor (NLI-IF), a protein whose function is unknown. The spatial expression was in neural regions at neurula stages, and localized to neural tissues, branchial arches, and pronephroi. The translational knockdown of dullard resulted in failure of neural tube development and structural deformation of the pronephric tubules, and the embryos consequently showed a reduction of head development. In the same screening method for the pronephros-specific genes, we isolated the XTRAP-gamma gene that codes translucent-associated protein, one of the subunit of the translocon-associated protein complex. XTRAP is one of the proteins proposed to aid in the translocation of nascent polypeptides into the lumen of the endothelium reticulum. XTRAP-gamma is expressed in pronephros tubules during pronephros development. The translational knockdown of XTRAP-gamma caused the suppression of tubulogenesis, the increase of Xlim-1 expression, and the decrease of Xpax-2 and Xwnt-4 expression. This translational knockdown of XTRAP-gamma also inhibited differentiation of the pronephros in animal caps by the treatment with activin and retinoic acid. These results showed that XTRAP-gamma plays an important role in the pronephric development. As another approach to clarify the molecular mechanisms of renal development, we investigated the role of Xenopus frizzled-8 ( Xfz8 ) in pronephros development to study the relation between Wnt signaling and nephric development. Translational inhibition of Xfz8 caused reduction in the staining of a duct-specific antibody and defects in pronephric tubule branching, while there was no effect on the expression of early pronephric maker genes in the duct region. Histologic analysis showed that the Xfz8-inhibited cells failed to form a normal epithelial structure. These results suggest that Xfz8 is involved in the formation of epithelium in the developing pronephric duct and tubules.

Architecture of the Ribosome–Channel Complex Derived from Native Membranes

The mammalian Sec61 complex forms a protein translocation channel whose function depends upon its interaction with the ribosome and with membrane proteins of the endoplasmic reticulum (ER). To study these interactions, we determined structures of "native" ribosome-channel complexes derived from ER membranes. We find that the ribosome is linked to the channel by seven connections, but the junction may still provide a path for domains of nascent membrane proteins to move into the cytoplasm. In addition, the native channel is significantly larger than a channel formed by the Sec61 complex, due to the presence of a second membrane protein. We identified this component as TRAP, the translocon-associated protein complex. TRAP interacts with Sec61 through its transmembrane domain and has a prominent lumenal domain. The presence of TRAP in the native channel indicates that it may play a general role in translocation. Crystal structures of two Sec61 homologues were used to model the channel. This analysis indicates that there are four Sec61 complexes and two TRAP molecules in each native channel. Thus, we suggest that a single Sec61 complex may form a conduit for translocating polypeptides, while three copies of Sec61 play a structural role or recruit accessory factors such as TRAP.

The role of XTRAP-gamma in Xenopus pronephros development

We isolated and characterized the Xenopus translocon-associated protein XTRAP-gamma, one of four subunits of the translocon-associated protein complex. TRAP has been proposed to aid the translocation of nascent polypeptides into the lumen of the endoplasmic reticulum, but this has not been demonstrated until now. XTRAP-gamma was specifically expressed in the pronephros tubules of Xenopus laevis from stage 25 during kidney development. Antisense morpholino oligonucleotide-mediated knockdown of XTRAP-gamma suppressed tubulogenesis and decreased expression of the pronephros marker genes Pax-2 and Wnt-4. XTRAP-gamma morpholinos also inhibited differentiation of the pronephros in activin/retinoic acid-treated animal caps. We conclude that XTRAP-gamma plays an important role in the process of pronephros differentiation.

Substrate-specific function of the translocon-associated protein complex during translocation across the ER membrane.

Although the transport of model proteins across the mammalian ER can be reconstituted with purified Sec61p complex, TRAM, and signal recognition particle receptor, some substrates, such as the prion protein (PrP), are inefficiently or improperly translocated using only these components. Here, we purify a factor needed for proper translocation of PrP and identify it as the translocon-associated protein (TRAP) complex. Surprisingly, TRAP also stimulates vectorial transport of many, but not all, other substrates in a manner influenced by their signal sequences. Comparative analyses of several natural signal sequences suggest that a dependence on TRAP for translocation is not due to any single physical parameter, such as hydrophobicity of the signal sequence. Instead, a functional property of the signal, efficiency of its post-targeting role in initiating substrate translocation, correlates inversely with TRAP dependence. Thus, maximal translocation independent of TRAP can only be achieved with a signal sequence, such as the one from prolactin, whose strong interaction with the translocon mediates translocon gating shortly after targeting. These results identify the TRAP complex as a functional component of the translocon and demonstrate that it acts in a substrate-specific manner to facilitate the initiation of protein translocation.

Translocon-associated protein TRAP delta and a novel TRAP-like protein are coordinately expressed with pro-opiomelanocortin in Xenopus intermediate pituitary.

In the intermediate pituitary gland of Xenopus laevis, the expression levels of the prohormone pro-opiomelanocortin (POMC) can be readily manipulated. When the animal is placed on a black background, the gene for POMC is actively transcribed, whereas on a white background the gene is virtually inactive. In this study, we characterized two genes whose transcript levels in the intermediate pituitary are regulated in coordination with that for POMC. One of these codes for a protein homologous to translocon-associated protein TRAP delta, a subunit of a transmembrane protein complex located at the site where nascent secretory proteins enter the endoplasmic reticulum (ER). Both Xenopus and mice were found to express an alternatively spliced transcript that gives rise to a previously unknown version of the TRAP delta protein. The product of the second gene is a novel and highly conserved protein with structural similarity to glycoprotein gp25L, a constituent of another translocon-associated protein complex. A database search revealed the existence of a novel family of gp25L-related proteins whose members occur throughout the animal kingdom. Together, our data imply that (i) the group of ER proteins surrounding translocating polypeptide chains may be far more complex than previously expected, and (ii) a number of the accessory components of the translocon participate in early steps of prohormone biosynthesis.