The membrane of the endoplasmic reticulum (ER) of nucleated human cells harbors the protein translocon, which facilitates membrane integration or translocation of almost every newly synthesized ...polypeptide targeted to organelles of the endo- and exocytotic pathway. The translocon comprises the polypeptide-conducting Sec61 channel and several additional proteins and complexes that are permanently or transiently associated with the heterotrimeric Sec61 complex. This ensemble of proteins facilitates ER targeting of precursor polypeptides, modification of precursor polypeptides in transit through the Sec61 complex, and Sec61 channel gating, i.e., dynamic regulation of the pore forming subunit to mediate precursor transport and calcium efflux. Recently, cryoelectron tomography of translocons in native ER membrane vesicles, derived from human cell lines or patient fibroblasts, and even intact cells has given unprecedented insights into the architecture and dynamics of the native translocon and the Sec61 channel. These structural data are discussed in light of different Sec61 channel activities including ribosome receptor function, membrane insertion, and translocation of newly synthesized polypeptides as well as the putative physiological roles of the Sec61 channel as a passive ER calcium leak channel. Furthermore, the structural insights into the Sec61 channel are incorporated into an overview and update on Sec61 channel-related diseases-the Sec61 channelopathies-and novel therapeutic concepts for their treatment.
Thapsigargin (TG) inhibits the sarco/endoplasmic reticulum Ca
2+
ATPase (SERCA) pump and, when applied acutely, it initiates a Ca
2+
mobilisation that begins with the loss of Ca
2+
from the ...endoplasmic reticulum (ER) and culminates with store-operated Ca
2+
entry (SOCE) from the extracellular space. Using the popular model cell line HEK-293, we quantified TG-induced changes in cytosolic and ER Ca
2+
levels using FURA-2 and the FRET-based ER Ca
2+
sensor D1ER, respectively. Our analysis predicts an ER Ca
2+
leak of 5–6 µM⋅s
−1
for the typical basal ER Ca
2+
level of 335–407 µM in HEK-293 cells. The resulting cytosolic Ca
2+
transients reached peak amplitudes of 0.6–1.0 µM in the absence of external Ca
2+
and were amplified by SOCE that amounted to 28–30 nM⋅s
−1
in 1 mM external Ca
2+
. Additionally, cytosolic Ca
2+
transients were shaped by a Ca
2+
clearance of 10–13 nM⋅s
−1
. Using puromycin (PURO), which enhances the ER Ca
2+
leak, we show that TG-induced cytosolic Ca
2+
transients are directly related to ER Ca
2+
levels and to the ER Ca
2+
leak. A one-compartment model incorporating ER Ca
2+
leak and cytosolic Ca
2+
clearance accounted satisfactorily for the basic features of TG-induced Ca
2+
transients and underpinned the rule that an increase in amplitude associated with shortening of TG-induced cytosolic Ca
2+
transients most likely reflects an increase in ER Ca
2+
leak.
In mammalian cells, one‐third of all polypeptides are integrated into the membrane or translocated into the lumen of the endoplasmic reticulum (ER) via the Sec61 channel. While the Sec61 complex ...facilitates ER import of most precursor polypeptides, the Sec61‐associated Sec62/Sec63 complex supports ER import in a substrate‐specific manner. So far, mainly posttranslationally imported precursors and the two cotranslationally imported precursors of ERj3 and prion protein were found to depend on the Sec62/Sec63 complex in vitro. Therefore, we determined the rules for engagement of Sec62/Sec63 in ER import in intact human cells using a recently established unbiased proteomics approach. In addition to confirming ERj3, we identified 22 novel Sec62/Sec63 substrates under these in vivo‐like conditions. As a common feature, those previously unknown substrates share signal peptides (SP) with comparatively longer but less hydrophobic hydrophobic region of SP and lower carboxy‐terminal region of SP (C‐region) polarity. Further analyses with four substrates, and ERj3 in particular, revealed the combination of a slowly gating SP and a downstream translocation‐disruptive positively charged cluster of amino acid residues as decisive for the Sec62/Sec63 requirement. In the case of ERj3, these features were found to be responsible for an additional immunoglobulin heavy‐chain binding protein (BiP) requirement and to correlate with sensitivity toward the Sec61‐channel inhibitor CAM741. Thus, the human Sec62/Sec63 complex may support Sec61‐channel opening for precursor polypeptides with slowly gating SPs by direct interaction with the cytosolic amino‐terminal peptide of Sec61α or via recruitment of BiP and its interaction with the ER‐lumenal loop 7 of Sec61α. These novel insights into the mechanism of human ER protein import contribute to our understanding of the etiology of SEC63‐linked polycystic liver disease.
Databases
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (http://www.ebi.ac.uk/pride/archive/projects/Identifiers) with the dataset identifiers: PXD008178, PXD011993, and PXD012078. Supplementary information was deposited at Mendeley Data (https://data.mendeley.com/datasets/6s5hn73jcv/2).
Sec61 is a membrane protein channel via which polypeptides are transported into the ER lumen or integrated into the membrane. The Sec61‐associated Sec62/Sec63 complex supports ER transport in a substate‐specific manner, but it is unclear how and which precursor polypeptides are recognised by this machinery. Here, Richard Zimmermann and co‐authors explored the mechanism of Sec62/Sec63‐driven ER import in human cells. They identified 36 precursors as Sec62/Sec63‐clients; 26 of which had signal peptides with longer but less hydrophobic H‐regions. Further analysis revealed that a slowly‐gating SP and the inhibitory effect of a positively charged cluster of amino acids are required for Sec62/Sec63 interaction.
The Mycobacterium ulcerans exotoxin, mycolactone, is an inhibitor of co-translational translocation via the Sec61 complex. Mycolactone has previously been shown to bind to, and alter the structure of ...the major translocon subunit Sec61α, and change its interaction with ribosome nascent chain complexes. In addition to its function in protein translocation into the ER, Sec61 also plays a key role in cellular Ca2+ homeostasis, acting as a leak channel between the endoplasmic reticulum (ER) and cytosol. Here, we have analysed the effect of mycolactone on cytosolic and ER Ca2+ levels using compartment-specific sensors. We also used molecular docking analysis to explore potential interaction sites for mycolactone on translocons in various states. These results show that mycolactone enhances the leak of Ca2+ ions via the Sec61 translocon, resulting in a slow but substantial depletion of ER Ca2+. This leak was dependent on mycolactone binding to Sec61α because resistance mutations in this protein completely ablated the increase. Molecular docking supports the existence of a mycolactone-binding transient inhibited state preceding translocation and suggests mycolactone may also bind Sec61α in its idle state. We propose that delayed ribosomal release after translation termination and/or translocon 'breathing' during rapid transitions between the idle and intermediate-inhibited states allow for transient Ca2+ leak, and mycolactone's stabilisation of the latter underpins the phenotype observed.
To fulfill its role in protein biogenesis, the endoplasmic reticulum (ER) depends on the Hsp70-type molecular chaperone BiP, which requires a constant ATP supply. However, the carrier that catalyzes ...ATP uptake into the ER was unknown. Here, we report that our screen of gene expression datasets for member(s) of the family of solute carriers that are co-expressed with BiP and are ER membrane proteins identifies SLC35B1 as a potential candidate. Heterologous expression of SLC35B1 in E. coli reveals that SLC35B1 is highly specific for ATP and ADP and acts in antiport mode. Moreover, depletion of SLC35B1 from HeLa cells reduces ER ATP levels and, as a consequence, BiP activity. Thus, human SLC35B1 may provide ATP to the ER and was named AXER (ATP/ADP exchanger in the ER membrane). Furthermore, we propose an ER to cytosol low energy response regulatory axis (termed lowER) that appears as central for maintaining ER ATP supply.
Eeyarestatin 1 (ES1) inhibits p97-dependent protein degradation, Sec61-dependent protein translocation into the endoplasmic reticulum (ER), and vesicular transport within the endomembrane system. ...Here, we show that ES1 impairs Ca
2+
homeostasis by enhancing the Ca
2+
leakage from mammalian ER. A comparison of various ES1 analogs suggested that the 5-nitrofuran (5-NF) ring of ES1 is crucial for this effect. Accordingly, the analog ES24, which conserves the 5-NF domain of ES1, selectively inhibited protein translocation into the ER, displayed the highest potency on ER Ca
2+
leakage of ES1 analogs studied and induced Ca
2+
-dependent cell death. Using small interfering RNA-mediated knockdown of Sec61α, we identified Sec61 complexes as the targets that mediate the gain of Ca
2+
leakage induced by ES1 and ES24. By interacting with the lateral gate of Sec61α, ES1 and ES24 likely capture Sec61 complexes in a Ca
2+
-permeable, open state, in which Sec61 complexes allow Ca
2+
leakage but are translocation incompetent.
•
ES1, ES2, and ES24 deplete Ca
2+
in ER
•
ESR35 and ES47 do not affect cellular Ca
2+
homeostasis
•
The most potent eeyarestatin, ES24, comprises only the 5-nitrofuran domain
•
ES1 and ES24 target Sec61 complexes in ER
Gamayun et al. discovered that eeyarestatins (ESs) interfere with closing mechanisms of Sec61 complexes of the ER and, as a “foot in the door,” stabilize Sec61 complexes in a Ca
2+
-permeable, open state. Specifically, ES24 enhances strongly the Sec61-mediated Ca
2+
leakage from ER and induces Ca
2+
-dependent cell death.
In mammalian cells, signal peptide‐dependent protein transport into the endoplasmic reticulum (ER) is mediated by a dynamic protein‐conducting channel, the Sec61 complex. Previous work has ...characterized the Sec61 channel as a potential ER Ca2+ leak channel and identified calmodulin as limiting Ca2+ leakage in a Ca2+‐dependent manner by binding to an IQ motif in the cytosolic aminoterminus of Sec61α. Here, we manipulated the concentration of the ER lumenal chaperone BiP in cells in different ways and used live cell Ca2+ imaging to monitor the effects of reduced levels of BiP on ER Ca2+ leakage. Regardless of how the BiP concentration was lowered, the absence of available BiP led to increased Ca2+ leakage via the Sec61 complex. When we replaced wild‐type Sec61α with mutant Sec61αY344H in the same model cell, however, Ca2+ leakage from the ER increased and was no longer affected by manipulation of the BiP concentration. Thus, BiP limits ER Ca2+ leakage through the Sec61 complex by binding to the ER lumenal loop 7 of Sec61α in the vicinity of tyrosine 344.
Sec61, the protein‐conducting channel of the endoplasmic reticulum, also mediates calcium efflux. The chaperone BiP binds to a luminal loop of Sec61 to inhibit calcium leakage and to link channel gating to protein homeostasis in the endoplasmic reticulum.
The ability to guide the growth of neurites is relevant for reconstructing neural networks and for nerve tissue regeneration. Here, a biofunctional hydrogel that allows light-based directional ...control of axon growth in situ is presented. The gel is covalently modified with a photoactivatable derivative of the short laminin peptidomimetic IKVAV. This adhesive peptide contains the photoremovable group 2-(4′-amino-4-nitro-1,1′-biphenyl-3-yl)propan-1-ol (HANBP) on the Lys rest that inhibits its activity. The modified peptide is highly soluble in water and can be simply conjugated to −COOH containing hydrogels via its terminal −NH2 group. Light exposure allows presentation of the IKVAV adhesive motif on a soft hydrogel at desired concentration and at defined position and time point. The photoactivated gel supports neurite outgrowth in embryonic neural progenitor cells culture and allows site-selective guidance of neurites extension. In situ exposure of cell cultures using a scanning laser allows outgrowth of neurites in desired pathways.
Protein transport into the endoplasmic reticulum (ER) is essential for all eukaryotic cells and evolutionary related to protein transport into and across the cytoplasmic membrane of eubacteria and ...archaea. It is based on amino-terminal signal peptides in the precursor polypeptides plus various transport components in cytosol plus ER and can occur either cotranslationally or posttranslationally. The two mechanisms merge at the heterotrimeric Sec61 complex in the ER membrane, which forms an aqueous polypeptide-conducting channel. Since the mammalian ER is also the main intracellular calcium storage organelle, the Sec61 complex is tightly regulated in its dynamics between the open and closed conformations by various ligands, such as precursor polypeptides at the cytosolic face and the Hsp70-type molecular chaperone BiP at the ER lumenal face (Hsp, heat shock protein). Furthermore, BiP binding to the incoming precursor polypeptide contributes to unidirectionality and efficiency of transport. Recent insights into the structural dynamics of the Sec61 complex and related complexes in eubacteria and archaea have various mechanistic and functional implications.
Display omitted
•The heterotrimeric Sec61 complex is present in the mammalian ER membrane.•It exists in a dynamic equilibrium between a closed conformation and an open conformation.•It can form an aqueous channel for the passage of thousands of different polypeptides.•In the open state, it also can allow passive calcium efflux from the ER.•Therefore, gating of the Sec61 channel is controlled by various allosteric effectors.
PDF
