Biogenesis Of Secretory Proteins Research Articles

Intramolecular quality control: HIV-1 envelope gp160 signal-peptide cleavage as a functional folding checkpoint.

Removal of the membrane-tethering signal peptides that target secretory proteins to the endoplasmic reticulum is a prerequisite for proper folding. While generally thought to be removed co-translationally, we report two additional post-targeting functions for the HIV-1 gp120 signal peptide, which remains attached until gp120 folding triggers its removal. First, the signal peptide improves folding fidelity by enhancing conformational plasticity of gp120 by driving disulfide isomerization through a redox-active cysteine. Simultaneously, the signal peptide delays folding by tethering the N terminus to the membrane, until assembly with the Cterminus. Second, its carefully timed cleavage represents intramolecular quality control and ensures release of (only) natively folded gp120. Postponed cleavage and the redox-active cysteine are both highly conserved and important for viral fitness. Considering the ∼15% proteins with signal peptides and the frequency of N-to-C contacts in protein structures, these regulatory roles of signal peptides are bound to be more common in secretory-protein biogenesis.

Ipomoeassin-F disrupts multiple aspects of secretory protein biogenesis

The Sec61 complex translocates nascent polypeptides into and across the membrane of the endoplasmic reticulum (ER), providing access to the secretory pathway. In this study, we show that Ipomoeassin-F (Ipom-F), a selective inhibitor of protein entry into the ER lumen, blocks the in vitro translocation of certain secretory proteins and ER lumenal folding factors whilst barely affecting others such as albumin. The effects of Ipom-F on protein secretion from HepG2 cells are twofold: reduced ER translocation combined, in some cases, with defective ER lumenal folding. This latter issue is most likely a consequence of Ipom-F preventing the cell from replenishing its ER lumenal chaperones. Ipom-F treatment results in two cellular stress responses: firstly, an upregulation of stress-inducible cytosolic chaperones, Hsp70 and Hsp90; secondly, an atypical unfolded protein response (UPR) linked to the Ipom-F-mediated perturbation of ER function. Hence, although levels of spliced XBP1 and CHOP mRNA and ATF4 protein increase with Ipom-F, the accompanying increase in the levels of ER lumenal BiP and GRP94 seen with tunicamycin are not observed. In short, although Ipom-F reduces the biosynthetic load of newly synthesised secretory proteins entering the ER lumen, its effects on the UPR preclude the cell restoring ER homeostasis.

Translational Control of Secretory Proteins in Health and Disease.

Secretory proteins are synthesized in a form of precursors with additional sequences at their N-terminal ends called signal peptides. The signal peptides are recognized co-translationally by signal recognition particle (SRP). This interaction leads to targeting to the endoplasmic reticulum (ER) membrane and translocation of the nascent chains into the ER lumen. It was demonstrated recently that in addition to a targeting function, SRP has a novel role in protection of secretory protein mRNAs from degradation. It was also found that the quality of secretory proteins is controlled by the recently discovered Regulation of Aberrant Protein Production (RAPP) pathway. RAPP monitors interactions of polypeptide nascent chains during their synthesis on the ribosomes and specifically degrades their mRNAs if these interactions are abolished due to mutations in the nascent chains or defects in the targeting factor. It was demonstrated that pathological RAPP activation is one of the molecular mechanisms of human diseases associated with defects in the secretory proteins. In this review, we discuss recent progress in understanding of translational control of secretory protein biogenesis on the ribosome and pathological consequences of its dysregulation in human diseases.

The ER exit sites are specialized ER zones for the transport of cargo proteins from the ER to the Golgi apparatus.

The endoplasmic reticulum (ER) is a multifunctional organelle, including secretory protein biogenesis, lipid synthesis, drug metabolism, Ca2+ signalling and so on. Since the ER is a single continuous membrane structure, it includes distinct zones responsible for its different functions. The export of newly synthesized proteins from the ER is facilitated via coat protein complex II (COPII)-coated vesicles, which form in specialized zones within the ER, called the ER exit sites (ERES) or transitional ER. In this review, we highlight recent advances in our understanding of the structural organization of ERES, the correlation between the ERES and Golgi organization, and the faithful cargo transport mechanism from the ERES to the Golgi.

Single particle trajectories reveal active endoplasmic reticulum luminal flow.

The Endoplasmic Reticulum (ER), a network of membranous sheets and pipes, supports functions encompassing biogenesis of secretory proteins and delivery of functional solutes throughout the cell1,2. Molecular mobility through the ER network enables these functionalities, but diffusion alone is not sufficient to explain luminal transport across supramicron distances. Understanding the ER structure-function relationship is critical in light of mutations in ER morphology regulating proteins that give rise to neurodegenerative disorders3,4. Here, super-resolution microscopy and analysis of single particle trajectories of ER luminal proteins revealed that the topological organization of the ER correlates with distinct trafficking modes of its luminal content: with a dominant diffusive component in tubular junctions and a fast flow component in tubules. Particle trajectory orientations resolved over time revealed an alternating current of the ER contents, whilst fast ER super-resolution identified energy-dependent tubule contraction events at specific points as a plausible mechanism for generating active ER luminal flow. The discovery of active flow in the ER has implications for timely ER content distribution throughout the cell, particularly important for cells with extensive ER-containing projections such as neurons.

Strukturelle Evolution des Signalerkennungspartikels

The signal recognition particle (SRP) is a ribonucleoprotein complex, which plays a central role in the first steps of membrane protein and secretory protein biogenesis. It recognizes nascent polypeptides at the ribosome by an N-terminal signal sequence and guides them to the translocation channel in the membrane of the endoplasmatic reticulum in eukaryotes, or the cell membrane in prokaryotes. Here, we focus on recent structural insights highlighting the SRP RNA as a dynamic module and on evolutionary aspects of RNA to protein transitions.

Defective secretory-protein mRNAs take the RAPP

Secretory proteins destined for the lumen of the endoplasmic reticulum are key regulators of cellular functions and are thus subject to several levels of quality control. A recent study finds that the earliest step in secretory protein biogenesis--binding of the signal recognition particle to the signal sequence of the nascent peptide--is subject to a quality control process termed RAPP for 'regulation of aberrant protein production'. This process involves AGO2 and mRNA degradation.

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

BackgroundThe 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.ResultsWe 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.ConclusionsWe 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.

Probing Endoplasmic Reticulum Dynamics using Fluorescence Imaging and Photobleaching Techniques

This unit describes approaches and tools for studying the dynamics and organization of endoplasmic reticulum (ER) membranes and proteins in living cells using fluorescence microscopy. The ER plays a key role in secretory protein biogenesis, calcium regulation, and lipid synthesis. However, study of these processes has often been restricted to biochemical assays that average millions of lysed cells or imaging of static fixed cells. With new fluorescent protein (FP) reporter tools, sensitive commercial microscopes, and photobleaching techniques, investigators can interrogate the behaviors of ER proteins, membranes, and stress pathways in single live cells. Solutions are described for imaging challenges relevant to the ER, including the mobility of ER membranes, a range of ER structures, and the influence of post-translational modifications on FP reporters. Considerations for performing photobleaching assays for ER proteins are discussed. Finally, reporters and drugs for studying misfolded secretory protein stress and the unfolded protein response are described.

Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway

The secretory pathway is responsible for the synthesis, folding, and delivery of a diverse array of cellular proteins. Secretory protein synthesis begins in the endoplasmic reticulum (ER), which is charged with the tasks of correctly integrating nascent proteins and ensuring correct post-translational modification and folding. Once ready for forward traffic, proteins are captured into ER-derived transport vesicles that form through the action of the COPII coat. COPII-coated vesicles are delivered to the early Golgi via distinct tethering and fusion machineries. Escaped ER residents and other cycling transport machinery components are returned to the ER via COPI-coated vesicles, which undergo similar tethering and fusion reactions. Ultimately, organelle structure, function, and cell homeostasis are maintained by modulating protein and lipid flux through the early secretory pathway. In the last decade, structural and mechanistic studies have added greatly to the strong foundation of yeast genetics on which this field was built. Here we discuss the key players that mediate secretory protein biogenesis and trafficking, highlighting recent advances that have deepened our understanding of the complexity of this conserved and essential process.

Unfolded Protein Responses With or Without Unfolded Proteins?

The endoplasmic reticulum (ER) is the site of secretory protein biogenesis. The ER quality control (QC) machinery, including chaperones, ensures the correct folding of secretory proteins. Mutant proteins and environmental stresses can overwhelm the available QC machinery. To prevent and resolve accumulation of misfolded secretory proteins in the ER, cells have evolved integral membrane sensors that orchestrate the Unfolded Protein Response (UPR). The sensors, Ire1p in yeast and IRE1, ATF6, and PERK in metazoans, bind the luminal ER chaperone BiP during homeostasis. As unfolded secretory proteins accumulate in the ER lumen, BiP releases, and the sensors activate. The mechanisms of activation and attenuation of the UPR sensors have exhibited unexpected complexity. A growing body of data supports a model in which Ire1p, and potentially IRE1, directly bind unfolded proteins as part of the activation process. However, evidence for an unfolded protein-independent mechanism has recently emerged, suggesting that UPR can be activated by multiple modes. Importantly, dysregulation of the UPR has been linked to human diseases including Type II diabetes, heart disease, and cancer. The existence of alternative regulatory pathways for UPR sensors raises the exciting possibility for the development of new classes of therapeutics for these medically important proteins.

Vacuolar H+-ATPase (V-ATPase) Promotes Vacuolar Membrane Permeabilization and Nonapoptotic Death in Stressed Yeast

Stress in the endoplasmic reticulum caused by tunicamycin, dithiothreitol, and azole-class antifungal drugs can induce nonapoptotic cell death in yeasts that can be blocked by the action of calcineurin (Cn), a Ca(2+)-dependent serine/threonine protein phosphatase. To identify additional factors that regulate nonapoptotic cell death in yeast, a collection of gene knock-out mutants was screened for mutants exhibiting altered survival rates. The screen revealed an endocytic protein (Ede1) that can function upstream of Ca(2+)/calmodulin-dependent protein kinase 2 (Cmk2) to suppress cell death in parallel to Cn. The screen also revealed the vacuolar H(+)-ATPase (V-ATPase), which acidifies the lysosome-like vacuole. The V-ATPase performed its death-promoting functions very soon after imposition of the stress and was not required for later stages of the cell death program. Cn did not inhibit V-ATPase activities but did block vacuole membrane permeabilization (VMP), which occurred at late stages of the cell death program. All of the other nondying mutants identified in the screens blocked steps before VMP. These findings suggest that VMP is the lethal event in dying yeast cells and that fungi may employ a mechanism of cell death similar to the necrosis-like cell death of degenerating neurons.

Genomewide Analysis Reveals Novel Pathways Affecting Endoplasmic Reticulum Homeostasis, Protein Modification and Quality Control

To gain new mechanistic insight into ER homeostasis and the biogenesis of secretory proteins, we screened a genomewide collection of yeast mutants for defective intracellular retention of the ER chaperone, Kar2p. We identified 87 Kar2p-secreting strains, including a number of known components in secretory protein modification and sorting. Further characterization of the 73 nonessential Kar2p retention mutants revealed roles for a number of novel gene products in protein glycosylation, GPI-anchor attachment, ER quality control, and retrieval of escaped ER residents. A subset of these mutants, required for ER retrieval, included the GET complex and two novel proteins that likely function similarly in membrane insertion of tail-anchored proteins. Finally, the variant histone, Htz1p, and its acetylation state seem to play an important role in maintaining ER retrieval pathways, suggesting a surprising link between chromatin remodeling and ER homeostasis.

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.

Sel1p/Ubx2p Participates in a Distinct Cdc48p‐Dependent Endoplasmic Reticulum‐Associated Degradation Pathway

The endoplasmic reticulum (ER) serves a critical role in the biogenesis of secretory proteins. Folding of nascent polypeptides occurs in the ER before anterograde transport through the secretory pathway, whereas terminally misfolded secretory proteins are recognized and eliminated by ER-associated degradation (ERAD). Here, we investigated the role of the ubiquitin regulatory X (UBX) domain-containing protein Sel1p in ER quality control and transport. Mutant sel1Delta yeast displayed a constitutively active unfolded protein response and a mildly reduced rate of secretory protein transport from the ER. Immunoisolation of Sel1p from detergent-solubilized ER microsomes revealed a protein complex containing both Cdc48p and Npl4p and suggested a direct role for Sel1p in ERAD. In cells that lack Sel1p, we observed a reduction in the level of Cdc48p bound to ER membranes and a decrease in the turnover rate of two model ERAD substrates, carboxypeptidase Y* and Ste6*. In addition, we found that Sel1p and a second UBX domain-containing protein, Shp1p, associated with Cdc48p in a mutually exclusive manner. Interestingly, the association of Sel1p with Cdc48p was regulated by ATP, while the interaction of Shp1p with Cdc48p was not influenced by ATP. Based on these findings, we conclude that Sel1p operates in the ERAD pathway by coupling Cdc48p to ER membranes and that Shp1p acts in a distinct Cdc48p-dependent protein degradation pathway.

Screening for mutants defective in secretory protein maturation and ER quality control

A genetic strategy devised to understand the physiology of the unfolded protein response serendipitously generated mutants affecting a broad spectrum of functions needed for secretory protein biogenesis and quality control. These included N- and O-linked glycosylation, glycosylphosphatidylinositol anchor biosynthesis and transfer, protein folding, protein trafficking, lumenal ionic homeostasis, ER quality control, and ER associated protein degradation. As these pathways are incompletely understood, the screen provides a simple method for their genetic dissection. This article describes methods for isolating novel mutants of these pathways and strategies for identifying corresponding genes.

How antigenic peptides are made to fit their groove

The immune system is able detect and eliminate virus-infected cells by recognition of viral-derived peptides that are displayed at the cell surface. The proteasome generates these peptides by degradation of viral proteins in the cytosol. The peptides are then transported into the lumen of the endoplasmic reticulum (ER), where they bind to the major histocompatibility (MHC) class I complex. Assembly with the appropriate peptide in the ER leads to transport of the loaded MHC class I complex to the cell surface.Antigenic peptides produced by proteasomal cleavage often have N-terminal extensions that need to be removed, by an as yet unidentified aminopeptidase, for efficient antigen presentation. An attractive hypothesis is that such peptides are trimmed to their optimal length when their C terminus is already bound to the MHC class I complex; thus, each peptide would be ‘custom-tailored’ for an optimal fit in the specific binding groove to which it is attached. Two recent papers provide data that support this custom-tailoring in situ.Komlosh and colleagues 1xA role for a novel luminal endoplasmic reticulum aminopeptidase in final trimming of 26S proteasome-generated major histocompatibility complex class I antigenic peptides. Komlosh et al. J. Biol. Chem. 2001; 276: 30050–30056Crossref | PubMed | Scopus (33)See all References1 found that an N-terminally extended peptide generated by proteasomal cleavage in vitro is processed to its optimal length in the presence of ER-derived microsomes. Processing was dependent on the activity of the transporter for antigenic peptides in the ER membrane, and on the presence of an MHC class I complex that can bind this specific peptide. In addition, N-terminal peptide trimming was inhibited by the metallo-aminopeptidase inhibitor 1,10-phenanthroline. Brouwenstijn et al. 2xMHC class I molecules can direct proteolytic cleavage of antigenic precursors in the endoplasmic reticulum. Brouwenstijn et al. Immunity. 2001; 15: 95–104Abstract | Full Text | Full Text PDF | PubMed | Scopus (51)See all References2 made similar observations, but also went one step further: they showed that the N-terminally extended peptides, as well as the trimmed peptides, are bound to the MHC class I complex. If trimming was inhibited chemically by the aminopeptidase inhibitor LMCK, or by depletion of ER lumenal contents, only extended peptides co-immunoprecipitated with the MHC class I complex.Taken together, these data suggest that a soluble aminopeptidase in the ER lumen is responsible for the trimming of MHC class I bound antigenic peptides. The ER lumen is the site of secretory protein biogenesis, and contains a high concentration of folding intermediates that are protease-sensitive. The activity of the trimming N-peptidase therefore must be strictly controlled to prevent it from chewing on proteins that are in the process of folding. This could be achieved by coupling the activity of the peptidase to its interaction with the loaded MHC class I complex or, perhaps, with tapasin – a specific chaperone involved in peptide loading. Purification and characterization of the trimming aminopeptidase will surely shed light on these questions.

How antigenic peptides are made to fit their groove

The immune system is able detect and eliminate virus-infected cells by recognition of viral-derived peptides that are displayed at the cell surface. The proteasome generates these peptides by degradation of viral proteins in the cytosol. The peptides are then transported into the lumen of the endoplasmic reticulum (ER), where they bind to the major histocompatibility (MHC) class I complex. Assembly with the appropriate peptide in the ER leads to transport of the loaded MHC class I complex to the cell surface.Antigenic peptides produced by proteasomal cleavage often have N-terminal extensions that need to be removed, by an as yet unidentified aminopeptidase, for efficient antigen presentation. An attractive hypothesis is that such peptides are trimmed to their optimal length when their C terminus is already bound to the MHC class I complex; thus, each peptide would be ‘custom-tailored’ for an optimal fit in the specific binding groove to which it is attached. Two recent papers provide data that support this custom-tailoring in situ.Komlosh and colleagues1xA role for a novel luminal endoplasmic reticulum aminopeptidase in final trimming of 26S proteasome-generated major histocompatibility complex class I antigenic peptides. Komlosh et al. J. Biol. Chem. 2001; 276: 30050–30056Crossref | PubMed | Scopus (31)See all References1 found that an N-terminally extended peptide generated by proteasomal cleavage in vitro is processed to its optimal length in the presence of ER-derived microsomes. Processing was dependent on the activity of the transporter for antigenic peptides in the ER membrane, and on the presence of an MHC class I complex that can bind this specific peptide. In addition, N-terminal peptide trimming was inhibited by the metallo-aminopeptidase inhibitor 1,10-phenanthroline. Brouwenstijn et al.2xMHC class I molecules can direct proteolytic cleavage of antigenic precursors in the endoplasmic reticulum. Brouwenstijn et al. Immunity. 2001; 15: 95–104Abstract | Full Text | Full Text PDF | PubMed | Scopus (50)See all References2 made similar observations, but also went one step further: they showed that the N-terminally extended peptides, as well as the trimmed peptides, are bound to the MHC class I complex. If trimming was inhibited chemically by the aminopeptidase inhibitor LMCK, or by depletion of ER lumenal contents, only extended peptides co-immunoprecipitated with the MHC class I complex.Taken together, these data suggest that a soluble aminopeptidase in the ER lumen is responsible for the trimming of MHC class I bound antigenic peptides. The ER lumen is the site of secretory protein biogenesis, and contains a high concentration of folding intermediates that are protease-sensitive. The activity of the trimming N-peptidase therefore must be strictly controlled to prevent it from chewing on proteins that are in the process of folding. This could be achieved by coupling the activity of the peptidase to its interaction with the loaded MHC class I complex or, perhaps, with tapasin – a specific chaperone involved in peptide loading. Purification and characterization of the trimming aminopeptidase will surely shed light on these questions.

Sec63p and Kar2p are required for the translocation of SRP-dependent precursors into the yeast endoplasmic reticulum in vivo.

The translocation of secretory polypeptides into the endoplasmic reticulum (ER) occurs at the translocon, a pore-forming structure that orchestrates the transport and maturation of polypeptides at the ER membrane. In yeast, targeting of secretory precursors to the translocon can occur by two distinct pathways that are distinguished by their dependence upon the signal recognition particle (SRP). The SRP-dependent pathway requires SRP and its membrane-bound receptor, whereas the SRP-independent pathway requires a separate receptor complex consisting of Sec62p, Sec63p, Sec71p, Sec72p plus lumenal Kar2p/BiP. Here we demonstrate that Sec63p and Kar2p are also required for the SRP-dependent targeting pathway in vivo. Furthermore, we demonstrate multiple roles for Sec63p, at least one of which is exclusive to the SRP-independent pathway.

Translocation of Proteins across the Endoplasmic Reticulum Membrane

Secretory protein biogenesis begins with the insertion of a preprotein into the lumen of the endoplasmic reticulum (ER). This insertion event, known as ER protein translocation, can occur either posttranslationally, in which the preprotein is completely synthesized on cytosolic ribosomes before being translocated, or cotranslationally, in which membrane-associated ribosomes direct the nascent polypeptide chain into the ER concomitant with polypeptide elongation. In either case, preproteins are targeted to the ER membrane through specific interactions with cytosolic and/or ER membrane factors. The preprotein is then transferred to a multiprotein translocation machine in the ER membrane that includes a pore through which the preprotein passes into the ER lumen. The energy required to drive protein translocation may derive either from the coupling of translation to translocation (during cotranslational translocation) or from ER lumenal molecular chaperones that may harness the preprotein or regulate the translocation machinery (during posttranslational translocation).