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Mechanisms of ER Protein Import

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Biochemistry".

Deadline for manuscript submissions: closed (30 June 2021) | Viewed by 53869

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Special Issue Editors

Prof. Dr. Richard Zimmermann

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Guest Editor

Medical Biochemistry and Molecular Biology, Medical Faculty, Saarland University, Homburg, Germany
Interests: human endoplasmic reticulum; protein import into the ER; calcium leakage from the ER; ATP/ADP exchange; molecular chaperones of the ER; Sec61 channel gating; Sec61-channelopathies; chaperonopathies

Dr. Sven Lang

E-Mail Website
Guest Editor

Medizinische Fakultät der Universität des Saarlandes, Universitätsklinikum des Saarlandes, Homburg, Germany
Interests: ATP transport; calcium homeostasis; calcium leakage; endoplasmic reticulum; membrane proteins; protein-protein interactions; protein targeting; protein transport; Sec61-channelopathies; Sec61 complex

Special Issue Information

Dear Colleagues,

Protein import into the endoplasmic reticulum (ER) is the first step in the biogenesis of approximately 10,000 different soluble and membrane proteins of mammalian cells, which amounts to about 30% of the proteome. All these proteins fulfill their functions either in the membrane or lumen of the ER itself, in one of the organelles of the pathways for endo- and exocytosis, or at the cell surface as plasma membrane or secreted proteins. ER protein import involves two stages—ER targeting, which guarantees membrane specificity, and the insertion of nascent membrane proteins into or translocation of soluble precursor polypeptides across the ER membrane. Typically, both processes depend on amino-terminal signal peptides or transmembrane helices. However, the targeting reaction may also involve the ER targeting of specific mRNAs. In addition, both processes may occur co- or post-translationally and are facilitated by various sophisticated machineries, which reside in the cytosol and the ER membrane, respectively. Except for resident ER proteins, proteins are delivered to their functional locations by vesicular transport. In this Special Issue, international experts in this area of cell biology report on their structural and mechanistic insights into various aspects of targeting, insertion, and translocation machineries, such as the signal recognition particle (SRP), its corresponding receptor, and the Sec61 complex. Furthermore, small-molecule inhibitors and toxins that interfere with ER protein import will be discussed in detail. With the last topic, this Special Issue provides a timely glimpse of how scientific analyses of ER protein import can contribute to the development of future therapeutic strategies against viruses, including SARS-CoV-2.

Prof. Dr. Richard Zimmermann
Dr. Sven Lang
Guest Editors

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Keywords

Protein targeting to the ER
Insertion of proteins into the ER membrane
Translocation of proteins into the ER
Signal peptides
Transmembrane helices
Intrinsically disordered domains
ER-SURF
SRP/SR
GET
SND
NAC
Sec61 complex
Sec61 translocon
EMC
PEX3
TRAP complex
Oligosaccharyltransferase
Signal peptidase
Sec61 channel inhibitors

Published Papers (13 papers)

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Editorial

Jump to: Research, Review

3 pages, 182 KiB

Open AccessEditorial

Mechanisms of ER Protein Import

by Sven Lang and Richard Zimmermann

Int. J. Mol. Sci. 2022, 23(10), 5315; https://doi.org/10.3390/ijms23105315 - 10 May 2022

Cited by 1 | Viewed by 1236

Abstract

Protein import into the endoplasmic reticulum (ER) is the first step in the biogenesis of approximately 10,000 different soluble and membrane proteins of human cells, which amounts to about 30% of the proteome [...] Full article

(This article belongs to the Special Issue Mechanisms of ER Protein Import)

Research

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22 pages, 3023 KiB

Open AccessArticle

Reduced DNAJC3 Expression Affects Protein Translocation across the ER Membrane and Attenuates the Down-Modulating Effect of the Translocation Inhibitor Cyclotriazadisulfonamide

by Eva Pauwels, Becky Provinciael, Anita Camps, Enno Hartmann and Kurt Vermeire

Int. J. Mol. Sci. 2022, 23(2), 584; https://doi.org/10.3390/ijms23020584 - 06 Jan 2022

Cited by 6 | Viewed by 1947

Abstract

One of the reported substrates for the endoplasmic reticulum (ER) translocation inhibitor cyclotriazadisulfonamide (CADA) is DNAJC3, a chaperone of the unfolded protein response during ER stress. In this study, we investigated the impact of altered DNAJC3 protein levels on the inhibitory activity of CADA. By comparing WT DNAJC3 with a CADA-resistant DNAJC3 mutant, we observed the enhanced sensitivity of human CD4, PTK7 and ERLEC1 for CADA when DNAJC3 was expressed at high levels. Combined treatment of CADA with a proteasome inhibitor resulted in synergistic inhibition of protein translocation and in the rescue of a small preprotein fraction, which presumably corresponds to the CADA affected protein fraction that is stalled at the Sec61 translocon. We demonstrate that DNAJC3 enhances the protein translation of a reporter protein that is expressed downstream of the CADA-stalled substrate, suggesting that DNAJC3 promotes the clearance of the clogged translocon. We propose a model in which a reduced DNAJC3 level by CADA slows down the clearance of CADA-stalled substrates. This results in higher residual translocation into the ER lumen due to the longer dwelling time of the temporarily stalled substrates in the translocon. Thus, by directly reducing DNAJC3 protein levels, CADA attenuates its net down-modulating effect on its substrates. Full article

(This article belongs to the Special Issue Mechanisms of ER Protein Import)

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22 pages, 2647 KiB

Open AccessArticle

Quantitative Proteomics and Differential Protein Abundance Analysis after the Depletion of PEX3 from Human Cells Identifies Additional Aspects of Protein Targeting to the ER

by Richard Zimmermann, Sven Lang, Monika Lerner, Friedrich Förster, Duy Nguyen, Volkhard Helms and Bianca Schrul

Int. J. Mol. Sci. 2021, 22(23), 13028; https://doi.org/10.3390/ijms222313028 - 01 Dec 2021

Cited by 7 | Viewed by 3025

Abstract

Protein import into the endoplasmic reticulum (ER) is the first step in the biogenesis of around 10,000 different soluble and membrane proteins in humans. It involves the co- or post-translational targeting of precursor polypeptides to the ER, and their subsequent membrane insertion or translocation. So far, three pathways for the ER targeting of precursor polypeptides and four pathways for the ER targeting of mRNAs have been described. Typically, these pathways deliver their substrates to the Sec61 polypeptide-conducting channel in the ER membrane. Next, the precursor polypeptides are inserted into the ER membrane or translocated into the ER lumen, which may involve auxiliary translocation components, such as the TRAP and Sec62/Sec63 complexes, or auxiliary membrane protein insertases, such as EMC and the TMCO1 complex. Recently, the PEX19/PEX3-dependent pathway, which has a well-known function in targeting and inserting various peroxisomal membrane proteins into pre-existent peroxisomal membranes, was also found to act in the targeting and, putatively, insertion of monotopic hairpin proteins into the ER. These either remain in the ER as resident ER membrane proteins, or are pinched off from the ER as components of new lipid droplets. Therefore, the question arose as to whether this pathway may play a more general role in ER protein targeting, i.e., whether it represents a fourth pathway for the ER targeting of precursor polypeptides. Thus, we addressed the client spectrum of the PEX19/PEX3-dependent pathway in both PEX3-depleted HeLa cells and PEX3-deficient Zellweger patient fibroblasts by an established approach which involved the label-free quantitative mass spectrometry of the total proteome of depleted or deficient cells, as well as differential protein abundance analysis. The negatively affected proteins included twelve peroxisomal proteins and two hairpin proteins of the ER, thus confirming two previously identified classes of putative PEX19/PEX3 clients in human cells. Interestingly, fourteen collagen-related proteins with signal peptides or N-terminal transmembrane helices belonging to the secretory pathway were also negatively affected by PEX3 deficiency, which may suggest compromised collagen biogenesis as a hitherto-unknown contributor to organ failures in the respective Zellweger patients. Full article

(This article belongs to the Special Issue Mechanisms of ER Protein Import)

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22 pages, 3647 KiB

Open AccessArticle

Lights, Camera, Interaction: Studying Protein–Protein Interactions of the ER Protein Translocase in Living Cells

by Mark Sicking, Martin Jung and Sven Lang

Int. J. Mol. Sci. 2021, 22(19), 10358; https://doi.org/10.3390/ijms221910358 - 26 Sep 2021

Cited by 8 | Viewed by 2757

Abstract

Various landmark studies have revealed structures and functions of the Sec61/SecY complex in all domains of live demonstrating the conserved nature of this ancestral protein translocase. While the bacterial homolog of the Sec61 complex resides in the plasma membrane, the eukaryotic counterpart manages the transfer of precursor proteins into or across the membrane of the endoplasmic reticulum (ER). Sec61 complexes are accompanied by a set of dynamically recruited auxiliary proteins assisting the transport of certain precursor polypeptides. TRAP and Sec62/Sec63 are two auxiliary protein complexes in mammalian cells that have been characterized by structural and biochemical methods. Using these ER membrane protein complexes for our proof-of-concept study, we aimed to detect interactions of membrane proteins in living mammalian cells under physiological conditions. Bimolecular luminescence complementation and competition was used to demonstrate multiple protein–protein interactions of different topological layouts. In addition to the interaction of the soluble catalytic and regulatory subunits of the cytosolic protein kinase A, we detected interactions of ER membrane proteins that either belong to the same multimeric protein complex (intra-complex interactions: Sec61α–Sec61β, TRAPα–TRAPβ) or protein complexes in juxtaposition (inter-complex interactions: Sec61α–TRAPα, Sec61α–Sec63, and Sec61β–Sec63). In the process, we established further control elements like synthetic peptide complementation for expression profiling of fusion constructs and protease-mediated reporter degradation demonstrating the cytosolic localization of a reporter complementation. Ease of use and flexibility of the approach presented here will spur further research regarding the dynamics of protein–protein interactions in response to changing cellular conditions in living cells. Full article

(This article belongs to the Special Issue Mechanisms of ER Protein Import)

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Review

Jump to: Editorial, Research

16 pages, 1356 KiB

Open AccessReview

Targeting of Proteins for Translocation at the Endoplasmic Reticulum

by Martin R. Pool

Int. J. Mol. Sci. 2022, 23(7), 3773; https://doi.org/10.3390/ijms23073773 - 29 Mar 2022

Cited by 13 | Viewed by 5274

Abstract

The endoplasmic reticulum represents the gateway to the secretory pathway. Here, proteins destined for secretion, as well as soluble and membrane proteins that reside in the endomembrane system and plasma membrane, are triaged from proteins that will remain in the cytosol or be targeted to other cellular organelles. This process requires the faithful recognition of specific targeting signals and subsequent delivery mechanisms to then target them to the translocases present at the ER membrane, which can either translocate them into the ER lumen or insert them into the lipid bilayer. This review focuses on the current understanding of the first step in this process representing the targeting phase. Targeting is typically mediated by cleavable N-terminal hydrophobic signal sequences or internal membrane anchor sequences; these can either be captured co-translationally at the ribosome or recognised post-translationally and then delivered to the ER translocases. Location and features of the targeting sequence dictate which of several overlapping targeting pathway substrates will be used. Mutations in the targeting machinery or targeting signals can be linked to diseases. Full article

(This article belongs to the Special Issue Mechanisms of ER Protein Import)

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15 pages, 2381 KiB

Open AccessReview

Fidelity of Cotranslational Protein Targeting to the Endoplasmic Reticulum

by Hao-Hsuan Hsieh and Shu-ou Shan

Int. J. Mol. Sci. 2022, 23(1), 281; https://doi.org/10.3390/ijms23010281 - 28 Dec 2021

Cited by 12 | Viewed by 2789

Abstract

Fidelity of protein targeting is essential for the proper biogenesis and functioning of organelles. Unlike replication, transcription and translation processes, in which multiple mechanisms to recognize and reject noncognate substrates are established in energetic and molecular detail, the mechanisms by which cells achieve a high fidelity in protein localization remain incompletely understood. Signal recognition particle (SRP), a conserved pathway to mediate the localization of membrane and secretory proteins to the appropriate cellular membrane, provides a paradigm to understand the molecular basis of protein localization in the cell. In this chapter, we review recent progress in deciphering the molecular mechanisms and substrate selection of the mammalian SRP pathway, with an emphasis on the key role of the cotranslational chaperone NAC in preventing protein mistargeting to the ER and in ensuring the organelle specificity of protein localization. Full article

(This article belongs to the Special Issue Mechanisms of ER Protein Import)

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49 pages, 4086 KiB

Open AccessReview

The Molecular Biodiversity of Protein Targeting and Protein Transport Related to the Endoplasmic Reticulum

by Andrea Tirincsi, Mark Sicking, Drazena Hadzibeganovic, Sarah Haßdenteufel and Sven Lang

Int. J. Mol. Sci. 2022, 23(1), 143; https://doi.org/10.3390/ijms23010143 - 23 Dec 2021

Cited by 10 | Viewed by 5162

Abstract

Looking at the variety of the thousands of different polypeptides that have been focused on in the research on the endoplasmic reticulum from the last five decades taught us one humble lesson: no one size fits all. Cells use an impressive array of components to enable the safe transport of protein cargo from the cytosolic ribosomes to the endoplasmic reticulum. Safety during the transit is warranted by the interplay of cytosolic chaperones, membrane receptors, and protein translocases that together form functional networks and serve as protein targeting and translocation routes. While two targeting routes to the endoplasmic reticulum, SRP (signal recognition particle) and GET (guided entry of tail-anchored proteins), prefer targeting determinants at the N- and C-terminus of the cargo polypeptide, respectively, the recently discovered SND (SRP-independent) route seems to preferentially cater for cargos with non-generic targeting signals that are less hydrophobic or more distant from the termini. With an emphasis on targeting routes and protein translocases, we will discuss those functional networks that drive efficient protein topogenesis and shed light on their redundant and dynamic nature in health and disease. Full article

(This article belongs to the Special Issue Mechanisms of ER Protein Import)

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16 pages, 5317 KiB

Open AccessReview

Folding and Insertion of Transmembrane Helices at the ER

by Paul Whitley, Brayan Grau, James C. Gumbart, Luis Martínez-Gil and Ismael Mingarro

Int. J. Mol. Sci. 2021, 22(23), 12778; https://doi.org/10.3390/ijms222312778 - 26 Nov 2021

Cited by 5 | Viewed by 2758

Abstract

In eukaryotic cells, the endoplasmic reticulum (ER) is the entry point for newly synthesized proteins that are subsequently distributed to organelles of the endomembrane system. Some of these proteins are completely translocated into the lumen of the ER while others integrate stretches of amino acids into the greasy 30 Å wide interior of the ER membrane bilayer. It is generally accepted that to exist in this non-aqueous environment the majority of membrane integrated amino acids are primarily non-polar/hydrophobic and adopt an α-helical conformation. These stretches are typically around 20 amino acids long and are known as transmembrane (TM) helices. In this review, we will consider how transmembrane helices achieve membrane integration. We will address questions such as: Where do the stretches of amino acids fold into a helical conformation? What is/are the route/routes that these stretches take from synthesis at the ribosome to integration through the ER translocon? How do these stretches ‘know’ to integrate and in which orientation? How do marginally hydrophobic stretches of amino acids integrate and survive as transmembrane helices? Full article

(This article belongs to the Special Issue Mechanisms of ER Protein Import)

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14 pages, 2783 KiB

Open AccessReview

Emerging View on the Molecular Functions of Sec62 and Sec63 in Protein Translocation

by Sung-jun Jung and Hyun Kim

Int. J. Mol. Sci. 2021, 22(23), 12757; https://doi.org/10.3390/ijms222312757 - 25 Nov 2021

Cited by 12 | Viewed by 4562

Abstract

Most secreted and membrane proteins are targeted to and translocated across the endoplasmic reticulum (ER) membrane through the Sec61 protein-conducting channel. Evolutionarily conserved Sec62 and Sec63 associate with the Sec61 channel, forming the Sec complex and mediating translocation of a subset of proteins. For the last three decades, it has been thought that ER protein targeting and translocation occur via two distinct pathways: signal recognition particle (SRP)-dependent co-translational or SRP-independent, Sec62/Sec63 dependent post-translational translocation pathway. However, recent studies have suggested that ER protein targeting and translocation through the Sec translocon are more intricate than previously thought. This review summarizes the current understanding of the molecular functions of Sec62/Sec63 in ER protein translocation. Full article

(This article belongs to the Special Issue Mechanisms of ER Protein Import)

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21 pages, 5333 KiB

Open AccessReview

Inhibitors of the Sec61 Complex and Novel High Throughput Screening Strategies to Target the Protein Translocation Pathway

by Eva Pauwels, Ralf Schülein and Kurt Vermeire

Int. J. Mol. Sci. 2021, 22(21), 12007; https://doi.org/10.3390/ijms222112007 - 05 Nov 2021

Cited by 12 | Viewed by 6746

Abstract

Proteins targeted to the secretory pathway start their intracellular journey by being transported across biological membranes such as the endoplasmic reticulum (ER). A central component in this protein translocation process across the ER is the Sec61 translocon complex, which is only intracellularly expressed and does not have any enzymatic activity. In addition, Sec61 translocon complexes are difficult to purify and to reconstitute. Screening for small molecule inhibitors impairing its function has thus been notoriously difficult. However, such translocation inhibitors may not only be valuable tools for cell biology, but may also represent novel anticancer drugs, given that cancer cells heavily depend on efficient protein translocation into the ER to support their fast growth. In this review, different inhibitors of protein translocation will be discussed, and their specific mode of action will be compared. In addition, recently published screening strategies for small molecule inhibitors targeting the whole SRP-Sec61 targeting/translocation pathway will be summarized. Of note, slightly modified assays may be used in the future to screen for substances affecting SecYEG, the bacterial ortholog of the Sec61 complex, in order to identify novel antibiotic drugs. Full article

(This article belongs to the Special Issue Mechanisms of ER Protein Import)

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19 pages, 2924 KiB

Open AccessReview

Take Me Home, Protein Roads: Structural Insights into Signal Peptide Interactions during ER Translocation

by A. Manuel Liaci and Friedrich Förster

Int. J. Mol. Sci. 2021, 22(21), 11871; https://doi.org/10.3390/ijms222111871 - 01 Nov 2021

Cited by 24 | Viewed by 7466

Abstract

Cleavable endoplasmic reticulum (ER) signal peptides (SPs) and other non-cleavable signal sequences target roughly a quarter of the human proteome to the ER. These short peptides, mostly located at the N-termini of proteins, are highly diverse. For most proteins targeted to the ER, it is the interactions between the signal sequences and the various ER targeting and translocation machineries such as the signal recognition particle (SRP), the protein-conducting channel Sec61, and the signal peptidase complex (SPC) that determine the proteins’ target location and provide translocation fidelity. In this review, we follow the signal peptide into the ER and discuss the recent insights that structural biology has provided on the governing principles of those interactions. Full article

(This article belongs to the Special Issue Mechanisms of ER Protein Import)

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12 pages, 1331 KiB

Open AccessReview

Molecular Modeling of Signal Peptide Recognition by Eukaryotic Sec Complexes

by Pratiti Bhadra and Volkhard Helms

Int. J. Mol. Sci. 2021, 22(19), 10705; https://doi.org/10.3390/ijms221910705 - 02 Oct 2021

Cited by 7 | Viewed by 2600

Abstract

Here, we review recent molecular modelling and simulation studies of the Sec translocon, the primary component/channel of protein translocation into the endoplasmic reticulum (ER) and bacterial periplasm, respectively. Our focus is placed on the eukaryotic Sec61, but we also mention modelling studies on prokaryotic SecY since both systems operate in related ways. Cryo-EM structures are now available for different conformational states of the Sec61 complex, ranging from the idle or closed state over an inhibited state with the inhibitor mycolactone bound near the lateral gate, up to a translocating state with bound substrate peptide in the translocation pore. For all these states, computational studies have addressed the conformational dynamics of the translocon with respect to the pore ring, the plug region, and the lateral gate. Also, molecular simulations are addressing mechanistic issues of insertion into the ER membrane vs. translocation into the ER, how signal-peptides are recognised at all in the translocation pore, and how accessory proteins affect the Sec61 conformation in the co- and post-translational pathways. Full article

(This article belongs to the Special Issue Mechanisms of ER Protein Import)

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13 pages, 1281 KiB

Open AccessReview

ER-SURF: Riding the Endoplasmic Reticulum Surface to Mitochondria

by Christian Koch, Maya Schuldiner and Johannes M. Herrmann

Int. J. Mol. Sci. 2021, 22(17), 9655; https://doi.org/10.3390/ijms22179655 - 06 Sep 2021

Cited by 15 | Viewed by 5712

Abstract

Most mitochondrial proteins are synthesized in the cytosol and targeted to the mitochondrial surface in a post-translational manner. The surface of the endoplasmic reticulum (ER) plays an active role in this targeting reaction. ER-associated chaperones interact with certain mitochondrial membrane protein precursors and transfer them onto receptor proteins of the mitochondrial surface in a process termed ER-SURF. ATP-driven proteins in the membranes of mitochondria (Msp1, ATAD1) and the ER (Spf1, P5A-ATPase) serve as extractors for the removal of mislocalized proteins. If the re-routing to mitochondria fails, precursors can be degraded by ER or mitochondria-associated degradation (ERAD or MAD respectively) in a proteasome-mediated reaction. This review summarizes the current knowledge about the cooperation of the ER and mitochondria in the targeting and quality control of mitochondrial precursor proteins. Full article

(This article belongs to the Special Issue Mechanisms of ER Protein Import)

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