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1
Witham, Christopher M., Aleshanee L. Paxman, Lamprini Baklous, Robert F. L. Steuart, Benjamin L. Schulz, and Carl J. Mousley. "Cancer associated mutations in Sec61γ alter the permeability of the ER translocase." PLOS Genetics 17, no. 8 (August 30, 2021): e1009780. http://dx.doi.org/10.1371/journal.pgen.1009780.
Full textAbstract:
Translocation of secretory and integral membrane proteins across or into the ER membrane occurs via the Sec61 complex, a heterotrimeric protein complex possessing two essential sub-units, Sec61p/Sec61α and Sss1p/Sec61γ and the non-essential Sbh1p/Sec61β subunit. In addition to forming a protein conducting channel, the Sec61 complex maintains the ER permeability barrier, preventing flow of molecules and ions. Loss of Sec61 integrity is detrimental and implicated in the progression of disease. The Sss1p/Sec61γ C-terminus is juxtaposed to the key gating module of Sec61p/Sec61α and is important for gating the translocon. Inspection of the cancer genome database identifies six mutations in highly conserved amino acids of Sec61γ/Sss1p. We identify that five out of the six mutations identified affect gating of the ER translocon, albeit with varying strength. Together, we find that mutations in Sec61γ that arise in malignant cells result in altered translocon gating dynamics, this offers the potential for the translocon to represent a target in co-therapy for cancer treatment.
2
Sicking, Mark, Martin Jung, and Sven Lang. "Lights, Camera, Interaction: Studying Protein–Protein Interactions of the ER Protein Translocase in Living Cells." International Journal of Molecular Sciences 22, no. 19 (September 26, 2021): 10358. http://dx.doi.org/10.3390/ijms221910358.
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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.
3
Witham, Christopher M., Hasindu G. Dassanayake, Aleshanee L. Paxman, Kofi L. P. Stevens, Lamprini Baklous, Paris F. White, Amy L. Black, et al. "The conserved C-terminus of Sss1p is required to maintain the endoplasmic reticulum permeability barrier." Journal of Biological Chemistry 295, no. 7 (December 17, 2019): 2125–34. http://dx.doi.org/10.1074/jbc.ra119.010378.
Full textAbstract:
The endoplasmic reticulum (ER) is the entry point to the secretory pathway and major site of protein biogenesis. Translocation of secretory and integral membrane proteins across or into the ER membrane occurs via the evolutionarily conserved Sec61 complex, a heterotrimeric channel that comprises the Sec61p/Sec61α, Sss1p/Sec61γ, and Sbh1p/Sec61β subunits. In addition to forming a protein-conducting channel, the Sec61 complex also functions to maintain the ER permeability barrier, preventing the mass free flow of essential ER-enriched molecules and ions. Loss in Sec61 integrity is detrimental and implicated in the progression of disease. The Sss1p/Sec61γ C terminus is juxtaposed to the key gating module of Sec61p/Sec61α, and we hypothesize it is important for gating the ER translocon. The ER stress response was found to be constitutively induced in two temperature-sensitive sss1 mutants (sss1ts) that are still proficient to conduct ER translocation. A screen to identify intergenic mutations that allow for sss1ts cells to grow at 37 °C suggests the ER permeability barrier to be compromised in these mutants. We propose the extreme C terminus of Sss1p/Sec61γ is an essential component of the gating module of the ER translocase and is required to maintain the ER permeability barrier.
4
Itskanov, Samuel, and Eunyong Park. "Structure of the posttranslational Sec protein-translocation channel complex from yeast." Science 363, no. 6422 (December 13, 2018): 84–87. http://dx.doi.org/10.1126/science.aav6740.
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The Sec61 protein-conducting channel mediates transport of many proteins, such as secretory proteins, across the endoplasmic reticulum (ER) membrane during or after translation. Posttranslational transport is enabled by two additional membrane proteins associated with the channel, Sec63 and Sec62, but its mechanism is poorly understood. We determined a structure of the Sec complex (Sec61-Sec63-Sec71-Sec72) from Saccharomyces cerevisiae by cryo–electron microscopy (cryo-EM). The structure shows that Sec63 tightly associates with Sec61 through interactions in cytosolic, transmembrane, and ER-luminal domains, prying open Sec61’s lateral gate and translocation pore and thus activating the channel for substrate engagement. Furthermore, Sec63 optimally positions binding sites for cytosolic and luminal chaperones in the complex to enable efficient polypeptide translocation. Our study provides mechanistic insights into eukaryotic posttranslational protein translocation.
5
KNIGHT, Bruce C., and Stephen HIGH. "Membrane integration of Sec61α: a core component of the endoplasmic reticulum translocation complex." Biochemical Journal 331, no. 1 (April 1, 1998): 161–67. http://dx.doi.org/10.1042/bj3310161.
Full textAbstract:
The Sec61 complex is a central component of the endoplasmic reticulum (ER) translocation site. The complex consists of three subunits: Sec61α, Sec61β and Sec61γ, at least two of which (α and β) are adjacent to nascent proteins during membrane insertion. Another component of the translocation machinery is the translocating chain-associating membrane (TRAM) protein, which is also adjacent to many nascent proteins during membrane insertion. Sec61α functions as the major component of a transmembrane channel formed by oligomers of the Sec61 complex. This channel is the site of secretory protein translocation and membrane protein integration at the ER membrane. Sec61α is a polytopic integral membrane protein, and we have studied its biosynthesis and membrane integration in vitro. Using a cross-linking approach to analyse the environment of a series of discrete Sec61α membrane-integration intermediates, we find: (i) newly synthesized Sec61α is adjacent to known components of the ER membrane-insertion site, namely Sec61α, Sec61β and TRAM, and thus the integration of Sec61α appears to require a pre-existing Sec61 complex; (ii) a site-specific cross-linking analysis indicates that the first transmembrane domain of Sec61α remains adjacent to protein components of the ER-insertion site (specifically TRAM and Sec61β) during the insertion of at least three subsequent transmembrane domains; and (iii) the membrane integration of Sec61α requires ER targeting by the signal-recognition particle.
6
Stirling, C. J., J. Rothblatt, M. Hosobuchi, R. Deshaies, and R. Schekman. "Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum." Molecular Biology of the Cell 3, no. 2 (February 1992): 129–42. http://dx.doi.org/10.1091/mbc.3.2.129.
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Yeast mutants defective in the translocation of soluble secretory proteins into the lumen of the endoplasmic reticulum (sec61, sec62, sec63) are not impaired in the assembly and glycosylation of the type II membrane protein dipeptidylaminopeptidase B (DPAPB) or of a chimeric membrane protein consisting of the multiple membrane-spanning domain of yeast hydroxymethylglutaryl CoA reductase (HMG1) fused to yeast histidinol dehydrogenase (HIS4C). This chimera is assembled in wild-type or mutant cells such that the His4c protein is oriented to the ER lumen and thus is not available for conversion of cytosolic histidinol to histidine. Cells harboring the chimera have been used to select new translocation defective sec mutants. Temperature-sensitive lethal mutations defining two complementation groups have been isolated: a new allele of sec61 and a single isolate of a new gene sec65. The new isolates are defective in the assembly of DPAPB, as well as the secretory protein alpha-factor precursor. Thus, the chimeric membrane protein allows the selection of more restrictive sec mutations rather than defining genes that are required only for membrane protein assembly. The SEC61 gene was cloned, sequenced, and used to raise polyclonal antiserum that detected the Sec61 protein. The gene encodes a 53-kDa protein with five to eight potential membrane-spanning domains, and Sec61p antiserum detects an integral protein localized to the endoplasmic reticulum membrane. Sec61p appears to play a crucial role in the insertion of secretory and membrane polypeptides into the endoplasmic reticulum.
7
Kalies, Kai-Uwe, Tom A. Rapoport, and Enno Hartmann. "The β Subunit of the Sec61 Complex Facilitates Cotranslational Protein Transport and Interacts with the Signal Peptidase during Translocation." Journal of Cell Biology 141, no. 4 (May 18, 1998): 887–94. http://dx.doi.org/10.1083/jcb.141.4.887.
Full textAbstract:
The Sec61 complex is the central component of the protein translocation apparatus of the ER membrane. We have addressed the role of the β subunit (Sec61β) during cotranslational protein translocation. With a reconstituted system, we show that a Sec61 complex lacking Sec61β is essentially inactive when elongation and membrane targeting of a nascent chain occur at the same time. The translocation process is perturbed at a step where the nascent chain would be inserted into the translocation channel. However, if sufficient time is given for the interaction of the nascent polypeptide with the mutant Sec61 complex, translocation is almost normal. Thus Sec61β kinetically facilitates cotranslational translocation, but is not essential for it. Using chemical cross-linking we show that Sec61β not only interacts with subunits of the Sec61 complex but also with the 25-kD subunit of the signal peptidase complex (SPC25), thus demonstrating for the first time a tight interaction between the SPC and the Sec61 complex. Interestingly, the cross-links between Sec61β and SPC25 and between Sec61β and Sec61α depend on the presence of membrane-bound ribosomes, suggesting that these interactions are induced when translocation is initiated. We propose that the SPC is transiently recruited to the translocation site, thus enhancing its activity.
8
Nelson, M. K., T. Kurihara, and P. A. Silver. "Extragenic suppressors of mutations in the cytoplasmic C terminus of SEC63 define five genes in Saccharomyces cerevisiae." Genetics 134, no. 1 (May 1, 1993): 159–73. http://dx.doi.org/10.1093/genetics/134.1.159.
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Abstract Mutations in the SEC63 gene of Saccharomyces cerevisiae affect both nuclear protein localization and translocation of proteins into the endoplasmic reticulum. We now report the isolation of suppressors of sec63-101 (formerly npl1-1), a temperature-sensitive allele of SEC63. Five complementation groups of extragenic mutations, son1-son5 (suppressor of npl1-1), were identified among the recessive suppressors. The son mutations are specific to SEC63, are not bypass suppressors, and are not new alleles of previously identified secretory (SEC61, SEC62, KAR2) or nuclear protein localization genes (NPL3, NPL4, NPL6). son1 mutations show regional specificity of suppression of sec63 alleles. At low temperatures, son1 mutants grow slowly and show partial mislocalization of nuclear antigens. The SON1 gene maps to chromosome IV and encodes a nuclear protein of 531 amino acids that contains two acidic stretches and a putative nuclear localization sequence. We show that son1 mutations suppress sec63-101 by elimination of Son1p function.
9
Baron, Ludivine, Anja Onerva Paatero, Jean-David Morel, Francis Impens, Laure Guenin-Macé, Sarah Saint-Auret, Nicolas Blanchard, et al. "Mycolactone subverts immunity by selectively blocking the Sec61 translocon." Journal of Experimental Medicine 213, no. 13 (November 7, 2016): 2885–96. http://dx.doi.org/10.1084/jem.20160662.
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Mycolactone, an immunosuppressive macrolide released by the human pathogen Mycobacterium ulcerans, was previously shown to impair Sec61-dependent protein translocation, but the underlying molecular mechanism was not identified. In this study, we show that mycolactone directly targets the α subunit of the Sec61 translocon to block the production of secreted and integral membrane proteins with high potency. We identify a single–amino acid mutation conferring resistance to mycolactone, which localizes its interaction site near the lumenal plug of Sec61α. Quantitative proteomics reveals that during T cell activation, mycolactone-mediated Sec61 blockade affects a selective subset of secretory proteins including key signal-transmitting receptors and adhesion molecules. Expression of mutant Sec61α in mycolactone-treated T cells rescued their homing potential and effector functions. Furthermore, when expressed in macrophages, the mycolactone-resistant mutant restored IFN-γ receptor–mediated antimicrobial responses. Thus, our data provide definitive genetic evidence that Sec61 is the host receptor mediating the diverse immunomodulatory effects of mycolactone and identify Sec61 as a novel regulator of immune cell functions.
10
Broughton, J., D. Swennen, B. M. Wilkinson, P. Joyet, C. Gaillardin, and C. J. Stirling. "Cloning of SEC61 homologues from Schizosaccharomyces pombe and Yarrowia lipolytica reveals the extent of functional conservation within this core component of the ER translocation machinery." Journal of Cell Science 110, no. 21 (November 1, 1997): 2715–27. http://dx.doi.org/10.1242/jcs.110.21.2715.
Full textAbstract:
The Sec61 protein is required for protein translocation across the ER membrane in both yeast and mammals and is found in close association with polypeptides during their membrane transit. In Saccharomyces cerevisiae Sec61p is essential for viability and the extent of sequence similarity between the yeast and mammalian proteins (55% sequence identity) suggests that the role of Sec61p in the translocation mechanism is likely to be conserved. In order to further our understanding of the structure and function of Sec61p we have cloned homologues from both Schizosaccharomyces pombe and Yarrowia lipolytica. The S. pombe gene comprises six exons encoding a 479 residue protein which we have immunolocalised to the endoplasmic reticulum. Sequence comparisons reveal that S. pombe Sec61p is 58.6% identical to that of S. cerevisiae. The deduced amino acid sequence of the Y. lipolytica protein shares 68.8% sequence identity with S. cerevisiae Sec61p. Gene disruption studies have shown that the SEC61 is required for viability in both S. pombe and Y. lipolytica demonstrating that the essential nature of this protein is not unique to S. cerevisiae. Moreover, heterologous complementation studies indicate that the Y. lipolytica SEC61 gene can complement a null mutation in S. cerevisiae. Sequence comparisons between the various eukaryotic Sec61p homologues reveal a number of highly conserved domains, including several transmembrane sequences and the majority of cytosolic loops. These comparisons will provide an important framework for the detailed analysis of interactions between Sec61p and other components of the translocation machinery and between Sec61p and translocating polypeptide chains.
11
Bhadra, Pratiti, Lalitha Yadhanapudi, Karin Römisch, and Volkhard Helms. "How does Sec63 affect the conformation of Sec61 in yeast?" PLOS Computational Biology 17, no. 3 (March 29, 2021): e1008855. http://dx.doi.org/10.1371/journal.pcbi.1008855.
Full textAbstract:
The Sec complex catalyzes the translocation of proteins of the secretory pathway into the endoplasmic reticulum and the integration of membrane proteins into the endoplasmic reticulum membrane. Some substrate peptides require the presence and involvement of accessory proteins such as Sec63. Recently, a structure of the Sec complex from Saccharomyces cerevisiae, consisting of the Sec61 channel and the Sec62, Sec63, Sec71 and Sec72 proteins was determined by cryo-electron microscopy (cryo-EM). Here, we show by co-precipitation that the Sec61 channel subunit Sbh1 is not required for formation of stable Sec63-Sec61 contacts. Molecular dynamics simulations started from the cryo-EM conformation of Sec61 bound to Sec63 and of unbound Sec61 revealed how Sec63 affects the conformation of Sec61 lateral gate, plug, pore region and pore ring diameter via three intermolecular contact regions. Molecular docking of SRP-dependent vs. SRP-independent signal peptide chains into the Sec61 channel showed that the pore regions affected by presence/absence of Sec63 play a crucial role in positioning the signal anchors of SRP-dependent substrates nearby the lateral gate.
12
Cheng, Zhiliang, Ying Jiang, Elisabet C. Mandon, and Reid Gilmore. "Identification of cytoplasmic residues of Sec61p involved in ribosome binding and cotranslational translocation." Journal of Cell Biology 168, no. 1 (January 3, 2005): 67–77. http://dx.doi.org/10.1083/jcb.200408188.
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The cytoplasmic surface of Sec61p is the binding site for the ribosome and has been proposed to interact with the signal recognition particle receptor during targeting of the ribosome nascent chain complex to the translocation channel. Point mutations in cytoplasmic loops six (L6) and eight (L8) of yeast Sec61p cause reductions in growth rates and defects in the translocation of nascent polypeptides that use the cotranslational translocation pathway. Sec61 heterotrimers isolated from the L8 sec61 mutants have a greatly reduced affinity for 80S ribosomes. Cytoplasmic accumulation of protein precursors demonstrates that the initial contact between the large ribosomal subunit and the Sec61 complex is important for efficient insertion of a nascent polypeptide into the translocation pore. In contrast, point mutations in L6 of Sec61p inhibit cotranslational translocation without significantly reducing the ribosome-binding activity, indicating that the L6 and L8 sec61 mutants affect different steps in the cotranslational translocation pathway.
13
Prinz, Anke, Enno Hartmann, and Kai-Uwe Kalies. "Sec61p Is the Main Ribosome Receptor in the Endoplasmic Reticulum of Saccharomyces cerevisiae." Biological Chemistry 381, no. 9-10 (September 13, 2000): 1025–28. http://dx.doi.org/10.1515/bc.2000.126.
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Abstract A characteristic feature of the co-translational protein translocation into the endoplasmic reticulum (ER) is the tight association of the translating ribosomes with the translocation sites in the membrane. Biochemical analyses identified the Sec61 complex as the main ribosome receptor in the ER of mammalian cells. Similar experiments using purified homologues from the yeast Saccharomyces cerevisiae, the Sec61p complex and the Ssh1p complex, respectively, demonstrated that they bind ribosomes with an affinity similar to that of the mammalian Sec61 complex. However, these studies did not exclude the presence of other proteins that may form abundant ribosome binding sites in the yeast ER. We now show here that similar to the situation found in mammals in the yeast Saccharomyces cerevisiae the two Sec61-homologues Sec61p and Ssh1p are essential for the formation of high-affinity ribosome binding sites in the ER membrane. The number of binding sites formed by Ssh1p under standard growth conditions is at least 4 times less than those formed by Sec61p.
14
Raden, David, Weiqun Song, and Reid Gilmore. "Role of the Cytoplasmic Segments of Sec61α in the Ribosome-Binding and Translocation-Promoting Activities of the Sec61 Complex." Journal of Cell Biology 150, no. 1 (July 10, 2000): 53–64. http://dx.doi.org/10.1083/jcb.150.1.53.
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The Sec61 complex performs a dual function in protein translocation across the RER, serving as both the high affinity ribosome receptor and the translocation channel. To define regions of the Sec61 complex that are involved in ribosome binding and translocation promotion, ribosome-stripped microsomes were subjected to limited digestions using proteases with different cleavage specificities. Protein immunoblot analysis using antibodies specific for the NH2 and COOH terminus of Sec61α was used to map the location of proteolysis cleavage sites. We observed a striking correlation between the loss of binding activity for nontranslating ribosomes and the digestion of the COOH- terminal tail or cytoplasmic loop 8 of Sec61α. The proteolyzed microsomes were assayed for SRP-independent translocation activity to determine whether high affinity binding of the ribosome to the Sec61 complex is a prerequisite for nascent chain transport. Microsomes that do not bind nontranslating ribosomes at physiological ionic strength remain active in SRP-independent translocation, indicating that the ribosome binding and translocation promotion activities of the Sec61 complex do not strictly correlate. Translocation-promoting activity was most severely inhibited by cleavage of cytosolic loop 6, indicating that this segment is a critical determinant for this function of the Sec61 complex.
15
Pilon, Marinus, Karin Römisch, Dong Quach, and Randy Schekman. "Sec61p Serves Multiple Roles in Secretory Precursor Binding and Translocation into the Endoplasmic Reticulum Membrane." Molecular Biology of the Cell 9, no. 12 (December 1998): 3455–73. http://dx.doi.org/10.1091/mbc.9.12.3455.
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The evolutionarily conserved Sec61 protein complex mediates the translocation of secretory proteins into the endoplasmic reticulum. To investigate the role of Sec61p, which is the main subunit of this complex, we generated recessive, cold-sensitive alleles ofsec61 that encode stably expressed proteins with strong defects in translocation. The stage at which posttranslational translocation was blocked was probed by chemical crosslinking of radiolabeled secretory precursors added to membranes isolated from wild-type and mutant strains. Two classes of sec61mutants were distinguished. The first class of mutants was defective in preprotein docking onto a receptor site of the translocon that included Sec61p itself. The second class of mutants allowed docking of precursors onto the translocon but was defective in the ATP-dependent release of precursors from this site that in wild-type membranes leads to pore insertion and full translocation. Only mutants of the second class were partially suppressed by overexpression ofSEC63, which encodes a subunit of the Sec61 holoenzyme complex responsible for positioning Kar2p (yeast BiP) at the translocation channel. These mutants thus define two early stages of translocation that require SEC61 function before precursor protein transfer across the endoplasmic reticulum membrane.
16
Green, N., H. Fang, and P. Walter. "Mutants in three novel complementation groups inhibit membrane protein insertion into and soluble protein translocation across the endoplasmic reticulum membrane of Saccharomyces cerevisiae." Journal of Cell Biology 116, no. 3 (February 1, 1992): 597–604. http://dx.doi.org/10.1083/jcb.116.3.597.
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We have isolated mutants that inhibit membrane protein insertion into the ER membrane of Saccharomyces cerevisiae. The mutants were contained in three complementation groups, which we have named SEC70, SEC71, and SEC72. The mutants also inhibited the translocation of soluble proteins into the lumen of the ER, indicating that they pleiotropically affect protein transport across and insertion into the ER membrane. Surprisingly, the mutants inhibited the translocation and insertion of different proteins to drastically different degrees. We have also shown that mutations in SEC61 and SEC63, which were previously isolated as mutants inhibiting the translocation of soluble proteins, also affect the insertion of membrane proteins into the ER. Taken together our data indicate that the process of protein translocation across the ER membrane involves a much larger number of gene products than previously appreciated. Moreover, different translocation substrates appear to have different requirements for components of the cellular targeting and translocation apparatus.
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Rothblatt, J. A., R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman. "Multiple genes are required for proper insertion of secretory proteins into the endoplasmic reticulum in yeast." Journal of Cell Biology 109, no. 6 (December 1, 1989): 2641–52. http://dx.doi.org/10.1083/jcb.109.6.2641.
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Genes that function in translocation of secretory protein precursors into the ER have been identified by a genetic selection for mutant yeast cells that fail to translocate a signal peptide-cytosolic enzyme hybrid protein. The new mutants, sec62 and sec63, are thermosensitive for growth and accumulate a variety of soluble secretory and vacuolar precursors whose electrophoretic mobilities coincide with those of the corresponding in vitro translated polypeptides. Proteolytic sensitivity of precursor molecules in extracts of mutant cells confirms that polypeptide translocation is blocked. Some form of interaction among the SEC61 (Deshaies, R. J., and R. Schekman. 1987. J. Cell Biol. 105:633-645), SEC62 and SEC63 gene products is suggested by the observation that haploid cells containing any pair of the mutations are inviable at 24 degrees C and show a marked enhancement of the translocation defect. The translocation defects of two mutants (sec62 and sec63) have been reproduced in vitro. sec63 microsomes display low and thermolabile translocation activity for prepro-alpha-factor (pp alpha F) synthesized with a cytosol fraction from wild type yeast. These gene products may constitute part of the polypeptide recognition or translocation apparatus of the ER membrane. Pulse-chase analysis of the translocation-defective mutants demonstrates that insertion of pp alpha F into the ER can proceed posttranslationally.
18
Kida, Yuichiro, and Masao Sakaguchi. "Interaction mapping of the Sec61 translocon identifies two Sec61α regions interacting with hydrophobic segments in translocating chains." Journal of Biological Chemistry 293, no. 44 (September 13, 2018): 17050–60. http://dx.doi.org/10.1074/jbc.ra118.003219.
Full textAbstract:
Many proteins in organelles of the secretory pathway, as well as secretory proteins, are translocated across and inserted into the endoplasmic reticulum membrane by the Sec61 translocon, a protein-conducting channel. The channel consists of 10 transmembrane (TM) segments of the Sec61α subunit and possesses an opening between TM2b and TM7, termed the lateral gate. Structural and biochemical analyses of complexes of Sec61 and its ortholog SecY have revealed that the lateral gate is the exit for signal sequences and TM segments of translocating polypeptides to the lipid bilayer and also involved in the recognition of such hydrophobic sequences. Moreover, even marginally hydrophobic (mH) segments insufficient for membrane integration can be transiently stalled in surrounding Sec61α regions and cross-linked to them, but how the Sec61 translocon accommodates these mH segments remains unclear. Here, we used Cys-scanned variants of human Sec61α expressed in cultured 293-H cells to examine which channel regions associate with mH segments. A TM segment in a ribosome-associated polypeptide was mainly cross-linked to positions at the lateral gate, whereas an mH segment in a nascent chain was cross-linked to the Sec61α pore-interior positions at TM5 and TM10, as well as the lateral gate. Of note, cross-linking at position 180 in TM5 of Sec61α was reduced by an I179A substitution. We therefore conclude that at least two Sec61α regions, the lateral gate and the pore-interior site around TM5, interact with mH segments and are involved in accommodating them.
19
Kalies, K. U., D. Görlich, and T. A. Rapoport. "Binding of ribosomes to the rough endoplasmic reticulum mediated by the Sec61p-complex." Journal of Cell Biology 126, no. 4 (August 15, 1994): 925–34. http://dx.doi.org/10.1083/jcb.126.4.925.
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The cotranslational translocation of proteins across the ER membrane involves the tight binding of translating ribosomes to the membrane, presumably to ribosome receptors. The identity of the latter has been controversial. One putative receptor candidate is Sec61 alpha, a multi-spanning membrane protein that is associated with two additional membrane proteins (Sec61 beta and gamma) to form the Sec61p-complex. Other receptors of 34 and 180 kD have also been proposed on the basis of their ability to bind at low salt concentration ribosomes lacking nascent chains. We now show that the Sec61p-complex has also binding activity but that, at low salt conditions, it accounts for only one third of the total binding sites in proteoliposomes reconstituted from a detergent extract of ER membranes. Under these conditions, the assay has also limited specificity with respect to ribosomes. However, if the ribosome-binding assay is performed at physiological salt concentration, most of the unspecific binding is lost; the Sec61p-complex then accounts for the majority of specific ribosome-binding sites in reconstituted ER membranes. To study the membrane interaction of ribosomes participating in protein translocation, native rough microsomes were treated with proteases. The amount of membrane-bound ribosomes is only slightly reduced by protease treatment, consistent with the protease-resistance of Sec61 alpha which is shielded by these ribosomes. In contrast, p34 and p180 can be readily degraded, indicating that they are not essential for the membrane anchoring of ribosomes in protease-treated microsomes. These data provide further evidence that the Sec61p-complex is responsible for the membrane-anchoring of ribosomes during translocation and make it unlikely that p34 or p180 are essential for this process.
20
Trueman, Steven F., Elisabet C. Mandon, and Reid Gilmore. "Translocation channel gating kinetics balances protein translocation efficiency with signal sequence recognition fidelity." Molecular Biology of the Cell 22, no. 17 (September 2011): 2983–93. http://dx.doi.org/10.1091/mbc.e11-01-0070.
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The transition between the closed and open conformations of the Sec61 complex permits nascent protein insertion into the translocation channel. A critical event in this structural transition is the opening of the lateral translocon gate that is formed by four transmembrane (TM) spans (TM2, TM3, TM7, and TM8 in Sec61p) to expose the signal sequence–binding site. To gain mechanistic insight into lateral gate opening, mutations were introduced into a lumenal loop (L7) that connects TM7 and TM8. The sec61 L7 mutants were found to have defects in both the posttranslational and cotranslational translocation pathways due to a kinetic delay in channel gating. The translocation defect caused by L7 mutations could be suppressed by the prl class of sec61 alleles, which reduce the fidelity of signal sequence recognition. The prl mutants are proposed to act by destabilizing the closed conformation of the translocation channel. Our results indicate that the equilibrium between the open and closed conformations of the protein translocation channel maintains a balance between translocation activity and signal sequence recognition fidelity.
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Plemper, R. K., J. Bordallo, P. M. Deak, C. Taxis, R. Hitt, and D. H. Wolf. "Genetic interactions of Hrd3p and Der3p/Hrd1p with Sec61p suggest a retro-translocation complex mediating protein transport for ER degradation." Journal of Cell Science 112, no. 22 (November 15, 1999): 4123–34. http://dx.doi.org/10.1242/jcs.112.22.4123.
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The endoplasmic reticulum contains a quality control system that subjects misfolded or unassembled secretory proteins to rapid degradation via the cytosolic ubiquitin proteasome system. This requires retrograde protein transport from the endoplasmic reticulum back to the cytosol. The Sec61 pore, the central component of the protein import channel into the endoplasmic reticulum, was identified as the core subunit of the retro-translocon as well. As import of mutated proteins into the endoplasmic reticulum lumen is successfully terminated, a new targeting mechanism must exist that mediates re-entering of misfolded proteins into the Sec61 pore from the lumenal side de novo. The previously identified proteins Der3p/Hrd1p and, as we show here, Hrd3p of the yeast Saccharomyces cerevisiae, are localised in the endoplasmic reticulum membrane and are essential for the degradation of several substrates of the endoplasmic reticulum degradation machinery. Based on genetic studies we demonstrate that they functionally interact with each other and with Sec61p, probably establishing the central part of the retro-translocon. In the absence of Hrd3p, the otherwise stable protein Der3p/Hrd1p becomes rapidly degraded. This depends on a functional ubiquitin proteasome system and the presence of substrate molecules of the endoplasmic reticulum degradation system. When overexpressed, Der3p/Hrd1p accelerates CPY* degradation in Delta(hrd3) cells. Our data suggest a recycling process of Der3p/Hrd1p through Hrd3p. The retro-translocon seems to be build up at least by the Sec61 pore, Der3p/Hrd1p and Hrd3p and mediates both retrograde transport and ubiquitination of substrate molecules.
22
Whang, Jennifer, Andrea Fan, Christopher Kirk, Eric Lowe, Dustin McMinn, Beatriz Millare, and Meera Rao. "207 Small molecule inhibitors of Sec61 cotranslational translocation regulate the phagocytosis checkpoint molecule CD47." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A223. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0207.
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BackgroundMany tumor cells escape immune cell clearance by overexpressing CD47, a multi-pass transmembrane protein, which binds signal regulatory protein α (SIRPα) on macrophages leading to decreased phagocytic activity. Blockade of CD47/SIRPα interactions enhances macrophage phagocytosis and is being targeted with antibody-based drugs, some of which are used in combination therapies in clinical trials. A novel method to target CD47 is through the inhibition of cotranslational translocation of transmembrane proteins. Immediately after exiting the ribosome, signal sequences that are unique to each protein are directed through the Sec61 channel into the ER for extracellular expression.1 Several Sec61-targeting compounds have been identified to suppress translocation in a signal sequence-specific manner.2 We previously described Sec61 inhibitors capable of selectively targeting immune checkpoint proteins and enhancing T cell function.3 Here, we demonstrate the blockade of CD47 expression on tumor cells and enhancement of macrophage phagocytosis with small molecule inhibitors of Sec61.MethodsSec61-dependent expression of target proteins was assayed using HEK293 cells overexpressing constructs comprised of signal sequences fused to a luciferase reporter. Stimulated PBMCs or tumor cells were incubated with Sec61 inhibitors, and surface expression of checkpoint molecules were examined by flow cytometry. Necrotic and apoptotic cells were assessed by Annexin V and 7AAD labeling. Human CD14+ monocytes were differentiated to M1- or M2-type macrophages. Jurkat or SKBR3 cells were incubated with Sec61 inhibitors, labeled with a pH sensitive dye and co-cultured with macrophages to assess phagocytosis.ResultsWe identified Sec61 inhibitors that block select immune checkpoint proteins. Compounds demonstrated either selective or multi-target profiles in transient transfection screens, which was supported by decreased protein expression on activated T cells. KZR-9275 targeted multiple checkpoint molecules, including PD-1, LAG-3 and CD73, along with a potent inhibition of the CD47 signal sequence reporter. CD47 surface expression was decreased on Jurkat and SKBR3 cells following 72 hours of compound treatment. KZR-9275 treatment of SKBR3 cells induced a minor increase in apoptotic cells, which was not detected in Jurkat cells. Increased macrophage phagocytosis, especially with M2-type macrophages, was observed when Jurkat or SKBR3 cells were pre-treated with KZR-9275.ConclusionsOur findings demonstrate that Sec61 inhibitors can block the expression of CD47, a phagocytosis checkpoint protein, on tumor cells and subsequently modulate macrophage phagocytic activity. Small molecule inhibitors of Sec61 provide an opportunity to target multiple checkpoint proteins on various cell populations. Future in vivo tumor models will assess the efficacy of Sec61 inhibitors to provide combination-like therapy.ReferencesPark E, Rapoport TA. Mechanisms of Sec61/SecY-mediated protein translocation across membranes. Annu Rev Biophys 2012; 41:1–20.Van Puyenbroeck V, Vermeire K. Inhibitors of protein translocation across membranes of the secretory pathway: novel antimicrobial and anticancer agents. Cell Mol Life Sci 2018; 75:1541–1558.Whang J, Anderl J, Fan A, Kirk C, Lowe E, McMinn D, et al. Targeting multiple immune checkpoint proteins with novel small molecule inhibitors of Sec61-dependent cotranslational translocation. 34th Annual Meeting & Pre-Conference Programs of the Society for Immunotherapy of Cancer (SITC 2019): part 2. J Immunother Cancer 2019; 7: 283. Abstract 815.
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Piña, Francisco J., Allyson F. O'Donnell, Silvere Pagant, Hai Lan Piao, John P. Miller, Stanley Fields, Elizabeth A. Miller, and Martha S. Cyert. "Hph1 and Hph2 Are Novel Components of the Sec63/Sec62 Posttranslational Translocation Complex That Aid in Vacuolar Proton ATPase Biogenesis." Eukaryotic Cell 10, no. 1 (November 19, 2010): 63–71. http://dx.doi.org/10.1128/ec.00241-10.
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ABSTRACT Hph1 and Hph2 are homologous integral endoplasmic reticulum (ER) membrane proteins required for Saccharomyces cerevisiae survival under environmental stress conditions. To investigate the molecular functions of Hph1 and Hph2, we carried out a split-ubiquitin-membrane-based yeast two-hybrid screen and identified their interactions with Sec71, a subunit of the Sec63/Sec62 complex, which mediates posttranslational translocation of proteins into the ER. Hph1 and Hph2 likely function in posttranslational translocation, as they interact with other Sec63/Sec62 complex subunits, i.e., Sec72, Sec62, and Sec63. hph1 Δ hph2 Δ cells display reduced vacuole acidification; increased instability of Vph1, a subunit of vacuolar proton ATPase (V-ATPase); and growth defects similar to those of mutants lacking V-ATPase activity. sec71 Δ cells exhibit similar phenotypes, indicating that Hph1/Hph2 and the Sec63/Sec62 complex function during V-ATPase biogenesis. Hph1/Hph2 and the Sec63/Sec62 complex may act together in this process, as vacuolar acidification and Vph1 stability are compromised to the same extent in hph1 Δ hph2 Δ and hph1 Δ hph2 Δ sec71 Δ cells. In contrast, loss of Pkr1, an ER protein that promotes posttranslocation assembly of Vph1 with V-ATPase subunits, further exacerbates hph1 Δ hph2 Δ phenotypes, suggesting that Hph1 and Hph2 function independently of Pkr1-mediated V-ATPase assembly. We propose that Hph1 and Hph2 aid Sec63/Sec62-mediated translocation of specific proteins, including factors that promote efficient biogenesis of V-ATPase, to support yeast cell survival during environmental stress.
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Greenfield, J. J., and S. High. "The Sec61 complex is located in both the ER and the ER-Golgi intermediate compartment." Journal of Cell Science 112, no. 10 (May 15, 1999): 1477–86. http://dx.doi.org/10.1242/jcs.112.10.1477.
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The heteromeric Sec61 complex is composed of (alpha), beta and (gamma) subunits and forms the core of the mammalian ER translocon. Oligomers of the Sec61 complex form a transmembrane channel where proteins are translocated across and integrated into the ER membrane. We have studied the subcellular localisation of the Sec61 complex using both wild-type COS1 cells and cells transfected with GFP-tagged Sec61(alpha). By double labelling immunofluorescence microscopy the GFP-tagged Sec61(alpha) was found in both the ER and the ER-Golgi intermediate compartment (ERGIC) but not in the trans-Golgi network. Immunofluorescence studies of endogenous Sec61beta and Sec61(gamma) showed that these proteins are also located in both the ER and the ERGIC. Using the alternative strategy of subcellular fractionation, we have shown that wild-type Sec61(alpha), beta and (gamma), and GFP-tagged Sec61(alpha), are all present in both the ER and the ERGIC/Golgi fractions of the gradient. The presence of the Sec61 subunits in a post-ER compartment suggests that these proteins can escape the ER and be recycled back, despite the fact that none of them contain any known membrane protein retrieval signals such as cytosolic di-lysine or di-arginine motifs. We also found that another translocon component, the glycoprotein TRAM, was present in post-ER compartments as demonstrated by subcellular fractionation. Our data indicate that the core components of the mammalian ER translocon are not permanently resident in the ER, but rather that they are maintained in the ER by a specific retrieval mechanism.
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Rubenstein, Eric M., Stefan G. Kreft, Wesley Greenblatt, Robert Swanson, and Mark Hochstrasser. "Aberrant substrate engagement of the ER translocon triggers degradation by the Hrd1 ubiquitin ligase." Journal of Cell Biology 197, no. 6 (June 11, 2012): 761–73. http://dx.doi.org/10.1083/jcb.201203061.
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Little is known about quality control of proteins that aberrantly or persistently engage the endoplasmic reticulum (ER)-localized translocon en route to membrane localization or the secretory pathway. Hrd1 and Doa10, the primary ubiquitin ligases that function in ER-associated degradation (ERAD) in yeast, target distinct subsets of misfolded or otherwise abnormal proteins based primarily on degradation signal (degron) location. We report the surprising observation that fusing Deg1, a cytoplasmic degron normally recognized by Doa10, to the Sec62 membrane protein rendered the protein a Hrd1 substrate. Hrd1-dependent degradation occurred when Deg1-Sec62 aberrantly engaged the Sec61 translocon channel and underwent topological rearrangement. Mutations that prevent translocon engagement caused a reversion to Doa10-dependent degradation. Similarly, a variant of apolipoprotein B, a protein known to be cotranslocationally targeted for proteasomal degradation, was also a Hrd1 substrate. Hrd1 therefore likely plays a general role in targeting proteins that persistently associate with and potentially obstruct the translocon.
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Kida, Yuichiro, Yudai Ishihara, Hidenobu Fujita, Yukiko Onishi, and Masao Sakaguchi. "Stability and flexibility of marginally hydrophobic–segment stalling at the endoplasmic reticulum translocon." Molecular Biology of the Cell 27, no. 6 (March 15, 2016): 930–40. http://dx.doi.org/10.1091/mbc.e15-09-0672.
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Many membrane proteins are integrated into the endoplasmic reticulum membrane through the protein-conducting channel, the translocon. Transmembrane segments with insufficient hydrophobicity for membrane integration are frequently found in multispanning membrane proteins, and such marginally hydrophobic (mH) segments should be accommodated, at least transiently, at the membrane. Here we investigated how mH-segments stall at the membrane and their stability. Our findings show that mH-segments can be retained at the membrane without moving into the lipid phase and that such segments flank Sec61α, the core channel of the translocon, in the translational intermediate state. The mH-segments are gradually transferred from the Sec61 channel to the lipid environment in a hydrophobicity-dependent manner, and this lateral movement may be affected by the ribosome. In addition, stalling mH-segments allow for insertion of the following transmembrane segment, forming an Ncytosol/Clumen orientation, suggesting that mH-segments can move laterally to accommodate the next transmembrane segment. These findings suggest that mH-segments may be accommodated at the ER membrane with lateral fluctuation between the Sec61 channel and the lipid phase.
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Grotzke, Jeff E., Patrycja Kozik, Jean-David Morel, Francis Impens, Natalia Pietrosemoli, Peter Cresswell, Sebastian Amigorena, and Caroline Demangel. "Sec61 blockade by mycolactone inhibits antigen cross-presentation independently of endosome-to-cytosol export." Proceedings of the National Academy of Sciences 114, no. 29 (July 5, 2017): E5910—E5919. http://dx.doi.org/10.1073/pnas.1705242114.
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Although antigen cross-presentation in dendritic cells (DCs) is critical to the initiation of most cytotoxic immune responses, the intracellular mechanisms and traffic pathways involved are still unclear. One of the most critical steps in this process, the export of internalized antigen to the cytosol, has been suggested to be mediated by Sec61. Sec61 is the channel that translocates signal peptide-bearing nascent polypeptides into the endoplasmic reticulum (ER), and it was also proposed to mediate protein retrotranslocation during ER-associated degradation (a process called ERAD). Here, we used a newly identified Sec61 blocker, mycolactone, to analyze Sec61’s contribution to antigen cross-presentation, ERAD, and transport of internalized antigens into the cytosol. As shown previously in other cell types, mycolactone prevented protein import into the ER of DCs. Mycolactone-mediated Sec61 blockade also potently suppressed both antigen cross-presentation and direct presentation of synthetic peptides to CD8+ T cells. In contrast, it did not affect protein export from the ER lumen or from endosomes into the cytosol, suggesting that the inhibition of cross-presentation was not related to either of these trafficking pathways. Proteomic profiling of mycolactone-exposed DCs showed that expression of mediators of antigen presentation, including MHC class I and β2 microglobulin, were highly susceptible to mycolactone treatment, indicating that Sec61 blockade affects antigen cross-presentation indirectly. Together, our data suggest that the defective translocation and subsequent degradation of Sec61 substrates is the cause of altered antigen cross-presentation in Sec61-blocked DCs.
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Cheng, Zhiliang. "Protein translocation through the Sec61/SecY channel." Bioscience Reports 30, no. 3 (February 17, 2010): 201–7. http://dx.doi.org/10.1042/bsr20090158.
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Special codes are embedded in the primary sequence of newly synthesized proteins to determine their final destination. Protein translocation across biological membranes requires co-operation between the targeting and translocation machineries. A conserved membrane channel, the Sec61/SecY complex, mediates protein translocation across or integration into the endoplasmic reticulum membrane in eukaryotes and the plasma membrane in prokaryotes. A combination of recent biochemical and structural data provides novel insights into the mechanism of how the channel allows polypeptide movement into the exoplasmic space and the lipid bilayer.
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Trueman, Steven F., Elisabet C. Mandon, and Reid Gilmore. "A gating motif in the translocation channel sets the hydrophobicity threshold for signal sequence function." Journal of Cell Biology 199, no. 6 (December 10, 2012): 907–18. http://dx.doi.org/10.1083/jcb.201207163.
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A critical event in protein translocation across the endoplasmic reticulum is the structural transition between the closed and open conformations of Sec61, the eukaryotic translocation channel. Channel opening allows signal sequence insertion into a gap between the N- and C-terminal halves of Sec61. We have identified a gating motif that regulates the transition between the closed and open channel conformations. Polar amino acid substitutions in the gating motif cause a gain-of-function phenotype that permits translocation of precursors with marginally hydrophobic signal sequences. In contrast, hydrophobic substitutions at certain residues in the gating motif cause a protein translocation defect. We conclude that the gating motif establishes the hydrophobicity threshold for functional insertion of a signal sequence into the Sec61 complex, thereby allowing the wild-type translocation channel to discriminate between authentic signal sequences and the less hydrophobic amino acid segments in cytosolic proteins. Bioinformatic analysis indicates that the gating motif is conserved between eubacterial and archaebacterial SecY and eukaryotic Sec61.
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Meyer, Lauren K., Cristina Delgado-Martin, Phillip P. Sharp, Dustin McMinn, Christopher J. Kirk, Jack Taunton, and Michelle L. Hermiston. "Protein Translocation Inhibitors Overcome Cytokine-Induced Glucocorticoid Resistance in T-Cell Acute Lymphoblastic Leukemia." Blood 134, Supplement_1 (November 13, 2019): 805. http://dx.doi.org/10.1182/blood-2019-127537.
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Glucocorticoids (GCs) are central to the treatment of T-cell acute lymphoblastic leukemia (T-ALL), and upfront resistance to GCs is a poor prognostic factor. We previously demonstrated that over one-third of primary patient T-ALLs are resistant to the GC dexamethasone (DEX) when cultured in the presence of interleukin-7 (IL7), a cytokine that is abundant in the microenvironment of leukemic blasts and that plays a well-established role in leukemogenesis. Mechanistically, we demonstrated that GCs paradoxically induce their own resistance by promoting the upregulation of IL7 receptor (IL7R) expression. In the presence of IL7, this augments signal transduction through the JAK/STAT5 axis, ultimately leading to increased STAT5 transcriptional output. This promotes the upregulation of the pro-survival protein BCL-2, which opposes DEX-induced apoptosis. Given that IL7-induced GC resistance depends on de novo synthesis of IL7R in response to DEX, and that newly synthesized IL7R reaches the cell surface via trafficking through the secretory pathway, we hypothesized that inhibiting the translocation of nascent IL7R peptide into the secretory pathway would effectively overcome IL7-induced DEX resistance. Sec61 is a protein-conducting channel in the membrane of the endoplasmic reticulum (ER) that is required for the cotranslational insertion of nascent polypeptides into the ER upon recognition of the signal sequence on secreted and cell surface proteins. To test the hypothesis that Sec61 inhibition could overcome IL7-induced DEX resistance, we utilized the human T-ALL cell line CCRF-CEM, which recapitulates the resistance phenotype observed in primary patient samples. Using a series of structurally distinct small molecule inhibitors of the Sec61 translocon, we demonstrated that Sec61 inhibition effectively overcomes the increase in cell surface IL7R expression in response to DEX. This occurs despite a persistent elevation in IL7R transcript expression following DEX exposure, confirming that Sec61 inhibitors act post-transcriptionally to attenuate cell surface IL7R expression. To determine whether the sensitivity of IL7R to Sec61 inhibitors is due specifically to the interaction between the IL7R signal sequence and Sec61 inhibitors, we generated IL7R constructs containing hydrophobic amino acid substitutions in the signal sequence, which are predicted to confer resistance to Sec61 inhibitors. Upon transient transfection of these constructs into HEK293T cells, we found that these mutations rendered IL7R resistant to the effects of Sec61 inhibition, confirming that the IL7R signal sequence confers sensitivity to these inhibitors. Using the Bliss independence model of synergy in CCRF-CEM cells, we demonstrated that Sec61 inhibitors potently synergize with DEX to overcome IL7-induced DEX resistance. Importantly, at concentrations at which synergy occurs, Sec61 inhibitors demonstrate no single-agent effect on cell survival, suggesting that these effects are not due to an overall reduction in secretory and membrane protein biogenesis. Furthermore, Sec61 inhibitors failed to sensitize CCRF-CEM cells to other chemotherapies used in T-ALL, none of which demonstrate IL7-induced resistance, thereby suggesting that these effects on DEX sensitivity are due specifically to the reduction in cell surface IL7R. To determine if Sec61 inhibitors prevent the DEX-induced increase in STAT5 transcription, we analyzed BCL-2 expression in cells exposed to DEX and IL7, and found that Sec61 inhibitors attenuate the increase in BCL-2 expression in a dose-dependent manner. We next analyzed a cohort of 34 primary patient T-ALL samples. As in CCRF-CEM cells, we found that specifically in those samples with IL7-induced DEX resistance, Sec61 inhibitors synergized with DEX to induce cell death in the presence of IL7. This effect occurred concomitantly with a reduction in cell surface IL7R expression and BCL-2 expression. Taken together, these data demonstrate the efficacy and feasibility of Sec61 inhibition as a novel and rational therapeutic strategy to overcome the IL7-induced DEX resistance phenotype that affects over one-third of newly diagnosed T-ALL patients. Disclosures Sharp: Kezar Life Sciences: Patents & Royalties. McMinn:Kezar Life Sciences: Employment, Equity Ownership. Kirk:Kezar Life Sciences: Employment, Equity Ownership. Taunton:Global Blood Therapeutics: Equity Ownership; Principia Biopharma: Equity Ownership, Patents & Royalties; Cedilla Therapeutics: Consultancy, Equity Ownership; Pfizer: Research Funding; Kezar Life Sciences: Equity Ownership, Patents & Royalties, Research Funding.
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Haßdenteufel, Sarah, Marie-Christine Klein, Armin Melnyk, and Richard Zimmermann. "Protein transport into the human ER and related diseases, Sec61-channelopathies." Biochemistry and Cell Biology 92, no. 6 (December 2014): 499–509. http://dx.doi.org/10.1139/bcb-2014-0043.
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Protein transport into the human endoplasmic reticulum (ER) is relevant to the biogenesis of most soluble and membrane proteins of organelles, which are involved in endo- or exo-cytsosis. It involves amino-terminal signal peptides in the precursor polypeptides and various transport components in the cytosol plus the ER, and can occur co- or post-translationally. The two mechanisms merge at the level of the ER membrane, specifically at the level of the heterotrimeric Sec61 complex, which forms a dynamic polypeptide-conducting channel in the ER membrane. Since the mammalian ER is also the main intracellular calcium storage organelle, and the Sec61 complex is calcium permeable, the Sec61 complex is tightly regulated in its equilibrium between the closed and open conformations, or “gated”, by ligands, such as signal peptides of the transport substrates and the ER lumenal Hsp70-type molecular chaperone BiP. Furthermore, BiP binding to the incoming polypeptide contributes to the efficiency and unidirectionality of transport. Recent insights into the structure and dynamic equilibrium of the Sec61 complex have various mechanistic as well as medical implications.
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Qian, Yu, Henry W. B. Johnson, Christopher J. Kirk, Eric Lowe, Dustin McMinn, Beatriz Millare, Tony Muchamuel, and Jinhai Wang. "Proteomic Profiling and Mechanistic Investigating of a Novel Anti-Cancer Small Molecule Inhibitor of Sec61." Blood 134, Supplement_1 (November 13, 2019): 2076. http://dx.doi.org/10.1182/blood-2019-125988.
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Secreted and transmembrane (TM) proteins play key roles in malignant transformation and tumor growth, including autocrine growth factor expression, receptor oncogene signal transduction pathways, metastasis, and immune system evasion. During translation, the majority of such proteins require translocation through the Sec61 translocon into the Endoplasmic Reticulum (ER) for further processing. This process is negotiated by unique signal sequences of the translating protein. Therefore, Sec61 represents a novel therapeutic target for cancer treatment through selective blockade of protein secretion. We generated Sec61 inhibitors and assessed their potential against target proteins using HEK293 cell lines stably expressing secreted or TM proteins of interest fused to a luciferase reporter. Additionally, anti-tumor activity was determined across both solid and liquid tumor cell lines in vitro and in mouse models. KZR-8834, a lead candidate identified through a medicinal chemistry campaign, induced cell death in multiple tumor cell lines in vitro, including multiple myeloma (MM), and was effective in xenograft models at doses that did not induce significant body weight loss or clinical signs of toxicity. We utilized quantitative proteomic methods to study KZR-8834 for inhibition of protein secretion and global modulation of protein homeostasis in sensitive and resistant tumor cell lines. Multiple tumor cell types were tested at various doses and time courses followed by subcellular fractionation of cytosolic and membrane/ER proteomes. Subsequent proteomic profiling was performed with Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) and/or Tandem Mass Tag 6-plex (TMT-sixplex). Sensitive targets from both proteomes were further verified using downstream biochemical methods. Sec61 client proteins showed both time- and dose-dependent inhibition upon compound treatment and proteomic results were verified via western blot analysis. Approximately 20% of the total Sec61 clientome and 25% of total proteins detected in a sensitive multiple myeloma (MM) cell line, H929, were significantly down-regulated in response to KZR-8834 treatment at concentrations leading to cell death. IPA pathway analysis suggested that activation of the ER stress response gene ATF4 was induced by KZR-8834 treatment in H929 cells. In a resistant MM cell line, U266, only 13% of the total Sec61 clientome and 5% of total protein detected were significantly down-regulated in response to the same compound treatment. A distinct profile of down-regulated Sec61 clientome was noted with overlap in only 11 of 394 commonly expressed proteins across those two cell lines. Interestingly, in compound treated cells, 39 down-regulated Sec61 client proteins in H929 were either unchanged or upregulated in U266 cells. Conversely, 38 upregulated H929 Sec61 clients were either unchanged or down-regulated in U266 cells. We further explored the ER stress response induced by KZR-8834 via comparative proteomic analysis in H929 cells treated with known ER stress inducers, Tunicamycin and Thapsigargin. These agents, which exert ER stress upon inhibition of N-linked glycosylation and blockade of ER Ca2+ flux, respectively, showed distinct cytosolic proteomic profiles in H929 cells relative to KZR-8834 treatment. These data suggest that KZR-8834-induced blockade of Sec61 results in a unique form of proteotoxic stress in sensitive MM cells. Collectively our results highlight quantitative proteomic profiling as a valuable tool toward elucidating the mechanism of pleiotropic acting molecules like KZR-8834. These studies constitute important first steps toward clarifying the anti-tumor mechanism inhibiting Sec61, a novel pathway agent, for the potential treatment of hematologic tumors. Disclosures Qian: Kezar Life Sciences: Employment, Equity Ownership. Johnson:Kezar Life Sciences: Employment, Equity Ownership. Kirk:Kezar Life Sciences: Employment, Equity Ownership. Lowe:Kezar Life Sciences: Employment, Equity Ownership. McMinn:Kezar Life Sciences: Employment, Equity Ownership. Millare:Kezar Life Sciences: Employment, Equity Ownership. Muchamuel:Kezar Life Sciences: Employment, Equity Ownership. Wang:Kezar Life Sciences: Employment, Equity Ownership.
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High, S., S. S. Andersen, D. Görlich, E. Hartmann, S. Prehn, T. A. Rapoport, and B. Dobberstein. "Sec61p is adjacent to nascent type I and type II signal-anchor proteins during their membrane insertion." Journal of Cell Biology 121, no. 4 (May 15, 1993): 743–50. http://dx.doi.org/10.1083/jcb.121.4.743.
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We have identified membrane components which are adjacent to type I and type II signal-anchor proteins during their insertion into the membrane of the ER. Using two different cross-linking approaches a 37-38-kD nonglycosylated protein, previously identified as P37 (High, S., D. Görlich, M. Wiedmann, T. A. Rapoport, and B. Dobberstein. 1991. J. Cell Biol. 113:35-44), was found adjacent to all the membrane inserted nascent chains used in this study. On the basis of immunoprecipitation, this ER protein was shown to be identical to the recently identified mammalian Sec61 protein. Thus, Sec61p is the principal cross-linking partner of both type I and type II signal-anchor proteins during their membrane insertion (this work), and of secretory proteins during their translocation (Görlich, D., S. Prehn, E. Hartmann, K.-U. Kalies, and T. A. Rapoport. 1992. Cell. 71:489-503). We propose that membrane proteins of both orientations, and secretory proteins employ the same ER translocation sites, and that Sec61p is a core component of these sites.
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Falcone, Domina, Matthew P. Henderson, Hendrik Nieuwland, Christine M. Coughlan, Jeffrey L. Brodsky, and David W. Andrews. "Stability and function of the Sec61 translocation complex depends on the Sss1p tail-anchor sequence." Biochemical Journal 436, no. 2 (May 13, 2011): 291–303. http://dx.doi.org/10.1042/bj20101865.
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Sss1p, an essential component of the heterotrimeric Sec61 complex in the ER (endoplasmic reticulum), is a tail-anchored protein whose precise mechanism of action is largely unknown. Tail-anchored proteins are involved in many cellular processes and are characterized by a single transmembrane sequence at or near the C-terminus. The Sec61 complex is the molecular machine through which secretory and membrane proteins translocate into and across the ER membrane. To understand the function of the tail anchor of Sss1p, we introduced mutations into the tail-anchor sequence and analysed the resulting yeast phenotypes. Point mutations in the C-terminal hydrophobic core of the tail anchor of Sss1p were identified that allowed Sss1p assembly into Sec61 complexes, but resulted in diminished growth, defects in co- and post-translational translocation, inefficient ribosome binding to Sec61 complexes, reduction in the stability of both heterotrimeric Sec61 and heptameric Sec complexes and a complete breakdown of ER structure. The underlying defect caused by the mutations involves loss of a stabilizing function of the Sss1p tail-anchor sequence for both the heterotrimeric Sec61 and the heptameric Sec complexes. These results indicate that by stabilizing multiprotein membrane complexes, the hydrophobic core of a tail-anchor sequence can be more than a simple membrane anchor.
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Deshaies, R. J., and R. Schekman. "A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum." Journal of Cell Biology 105, no. 2 (August 1, 1987): 633–45. http://dx.doi.org/10.1083/jcb.105.2.633.
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We have devised a genetic selection for mutant yeast cells that fail to translocate secretory protein precursors into the lumen of the endoplasmic reticulum (ER). Mutant cells are selected by a procedure that requires a signal peptide-containing cytoplasmic enzyme chimera to remain in contact with the cytosol. This approach has uncovered a new secretory mutant, sec61, that is thermosensitive for growth and that accumulates multiple secretory and vacuolar precursor proteins that have not acquired any detectable posttranslational modifications associated with translocation into the ER. Preproteins that accumulate at the sec61 block sediment with the particulate fraction, but are exposed to the cytosol as judged by sensitivity to proteinase K. Thus, the sec61 mutation defines a gene that is required for an early cytoplasmic or ER membrane-associated step in protein translocation.
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Sicking, Mark, Sven Lang, Florian Bochen, Andreas Roos, Joost P. H. Drenth, Muhammad Zakaria, Richard Zimmermann, and Maximilian Linxweiler. "Complexity and Specificity of Sec61-Channelopathies: Human Diseases Affecting Gating of the Sec61 Complex." Cells 10, no. 5 (April 27, 2021): 1036. http://dx.doi.org/10.3390/cells10051036.
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The rough endoplasmic reticulum (ER) of nucleated human cells has crucial functions in protein biogenesis, calcium (Ca2+) homeostasis, and signal transduction. Among the roughly one hundred components, which are involved in protein import and protein folding or assembly, two components stand out: The Sec61 complex and BiP. The Sec61 complex in the ER membrane represents the major entry point for precursor polypeptides into the membrane or lumen of the ER and provides a conduit for Ca2+ ions from the ER lumen to the cytosol. The second component, the Hsp70-type molecular chaperone immunoglobulin heavy chain binding protein, short BiP, plays central roles in protein folding and assembly (hence its name), protein import, cellular Ca2+ homeostasis, and various intracellular signal transduction pathways. For the purpose of this review, we focus on these two components, their relevant allosteric effectors and on the question of how their respective functional cycles are linked in order to reconcile the apparently contradictory features of the ER membrane, selective permeability for precursor polypeptides, and impermeability for Ca2+. The key issues are that the Sec61 complex exists in two conformations: An open and a closed state that are in a dynamic equilibrium with each other, and that BiP contributes to its gating in both directions in cooperation with different co-chaperones. While the open Sec61 complex forms an aqueous polypeptide-conducting- and transiently Ca2+-permeable channel, the closed complex is impermeable even to Ca2+. Therefore, we discuss the human hereditary and tumor diseases that are linked to Sec61 channel gating, termed Sec61-channelopathies, as disturbances of selective polypeptide-impermeability and/or aberrant Ca2+-permeability.
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Romisch, K. "Surfing the Sec61 channel: bidirectional protein translocation across the ER membrane." Journal of Cell Science 112, no. 23 (December 1, 1999): 4185–91. http://dx.doi.org/10.1242/jcs.112.23.4185.
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Misfolded secretory and transmembrane proteins are retained in the endoplasmic reticulum (ER) and subsequently degraded. Degradation is primarily mediated by cytosolic proteasomes and thus requires retrograde transport out of the ER back to the cytosol. The available evidence suggests that the protein-conducting channel formed by the Sec61 complex is responsible for both forward and retrograde transport of proteins across the ER membrane. For transmembrane proteins, retrograde transport can be viewed as a reversal of integration of membrane proteins into the ER membrane. Retrograde transport of soluble proteins through the Sec61 channel after signal-peptide cleavage, however, must be mechanistically distinct from signal-peptide-mediated import into the ER through the same channel.
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Junne, Tina, Torsten Schwede, Veit Goder, and Martin Spiess. "The Plug Domain of Yeast Sec61p Is Important for Efficient Protein Translocation, but Is Not Essential for Cell Viability." Molecular Biology of the Cell 17, no. 9 (September 2006): 4063–68. http://dx.doi.org/10.1091/mbc.e06-03-0200.
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The Sec61/SecY translocon mediates translocation of proteins across the membrane and integration of membrane proteins into the lipid bilayer. The structure of the translocon revealed a plug domain blocking the pore on the lumenal side. It was proposed to be important for gating the protein conducting channel and for maintaining the permeability barrier in its unoccupied state. Here, we analyzed in yeast the effect of introducing destabilizing point mutations in the plug domain or of its partial or complete deletion. Unexpectedly, even when the entire plug domain was deleted, cells were viable without growth phenotype. They showed an effect on signal sequence orientation of diagnostic signal-anchor proteins, a minor defect in cotranslational and a significant deficiency in posttranslational translocation. Steady-state levels of the mutant protein were reduced, and when coexpressed with wild-type Sec61p, the mutant lacking the plug competed poorly for complex partners. The results suggest that the plug is unlikely to be important for sealing the translocation pore in yeast but that it plays a role in stabilizing Sec61p during translocon formation.
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Lowe, Eric, Andrea R. Fan, Jing Jiang, Henry W. B. Johnson, Christopher J. Kirk, Dustin McMinn, Tony Muchamuel, Yu Qian, and Brian B. Tuch. "Blocking Protein Secretion with Novel Small Molecule Inhibitors of Sec61 Represents a Potential Treatment Strategy Against Hematologic Malignancies." Blood 134, Supplement_1 (November 13, 2019): 408. http://dx.doi.org/10.1182/blood-2019-123782.
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Secreted and membrane proteins play key roles in cancer development, including as autocrine growth factors, receptors in oncogenic signaling cascades, and checkpoints in immune system evasion. The biogenesis of most secreted and transmembrane proteins involves cotranslational translocation of nascent polypeptides ("clients") into the endoplasmic reticulum (ER) through the Sec61 translocon. Multiple inhibitors of protein secretion have been described that target Sec61 and have antitumor activity but lack adequate pharmaceutical properties or tolerability to be studied clinically. Here we describe novel small molecule inhibitors of Sec61 which exhibit activity against hematologic tumor cells in vitro and in vivo. KZR-8834 (8834) was identified through a screening campaign for novel inhibitors of Sec61-dependent protein secretion, where it exhibited nanomolar potency against multiple Sec61 client proteins of therapeutic value, including several immune checkpoint proteins. The broad Sec61 client inhibition profile of 8834 led to its in vitro assessment of anti-cancer activity against a panel of 346 human cancer cell lines. 8834 displayed broad cytotoxic activity against both solid and hematologic tumor types with high potency for hematologic malignancies including acute lymphoid leukemia (mean IC50=317nM, n=8 cell lines), acute myeloid leukemia (IC50=359nM, n=14), lymphoma (IC50=250nM, n=15) and multiple myeloma (IC50=352nM, n=11). In mouse xenograft models of multiple myeloma (H929) and mantle cell lymphoma (Mino), once weekly administration of 8834 or KZR-9261 (8834 analog) resulted in >90% tumor growth inhibition without significant clinical toxicity. Two multiple myeloma cell lines, H929 and U266, were chosen to assess cellular response due to their sensitivity and resistance to 8834, respectively. In H929 cells, 250nM 8834 induced activation of caspase 3/7 (>15 fold) within 8 hours of exposure which corresponded with a cell viability IC50 of 98nM at 24 hours. In contrast, no caspase 3/7 activation was noted in U266 cells through 24 hours of exposure, which corresponded with minimal effects on cell viability. Gene expression profiling by RNAseq and quantitative proteomic profiling by mass spectrometry was performed on these cell lines to elucidate mechanisms of sensitivity and resistance to Sec61 inhibition. Both methods revealed rapid upregulation of ER stress response genes/proteins and activation of the unfolded protein response, which was greater in H929 cells and confirmed by immunoblot and QPCR. Gene set enrichment analysis revealed significantly higher basal levels of ER stress-related genes in H929 vs U266 cells, suggesting ER stress response capacity as a possible predictive biomarker. In summary, blockade of Sec61-dependent translocation of secreted and membrane proteins with novel small molecule inhibitors exhibits a broad antitumor profile in vitro, potentially in part through activation of proteotoxic stress. These effects translate into therapeutic activity in multiple mouse xenograft models, demonstrating a potential novel treatment for hematologic malignancies. Disclosures Lowe: Kezar Life Sciences: Employment, Equity Ownership. Fan:Kezar Life Sciences: Employment, Equity Ownership. Jiang:Kezar Life Sciences: Employment, Equity Ownership. Johnson:Kezar Life Sciences: Employment, Equity Ownership. Kirk:Kezar Life Sciences: Employment, Equity Ownership. McMinn:Kezar Life Sciences: Employment, Equity Ownership. Muchamuel:Kezar Life Sciences: Employment, Equity Ownership. Qian:Kezar Life Sciences: Employment, Equity Ownership. Tuch:Kezar Life Sciences: Consultancy.
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Meacock, Suzanna L., Fabienne J. L. Lecomte, Samuel G. Crawshaw, and Stephen High. "Different Transmembrane Domains Associate with Distinct Endoplasmic Reticulum Components during Membrane Integration of a Polytopic Protein." Molecular Biology of the Cell 13, no. 12 (December 2002): 4114–29. http://dx.doi.org/10.1091/mbc.e02-04-0198.
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We have been studying the insertion of the seven transmembrane domain (TM) protein opsin to gain insights into how the multiple TMs of polytopic proteins are integrated at the endoplasmic reticulum (ER). We find that the ER components associated with the first and second TMs of the nascent opsin polypeptide chain are clearly distinct. The first TM (TM1) is adjacent to the α and β subunits of the Sec61 complex, and a novel component, a protein associated with the ER translocon of 10 kDa (PAT-10). The most striking characteristic of PAT-10 is that it remains adjacent to TM1 throughout the biogenesis and membrane integration of the full-length opsin polypeptide. TM2 is also found to be adjacent to Sec61α and Sec61β during its membrane integration. However, TM2 does not form any adducts with PAT-10; rather, a transient association with the TRAM protein is observed. We show that the association of PAT-10 with opsin TM1 does not require theN-glycosylation of the nascent chain and occurs irrespective of the amino acid sequence and transmembrane topology of TM1. We conclude that the precise makeup of the ER membrane insertion site can be distinct for the different transmembrane domains of a polytopic protein. We find that the environment of a particular TM can be influenced by both the “stage” of nascent chain biosynthesis reached, and the TM's relative location within the polypeptide.
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Osborne, Andrew R., Tom A. Rapoport, and Bert van den Berg. "PROTEIN TRANSLOCATION BY THE SEC61/SECY CHANNEL." Annual Review of Cell and Developmental Biology 21, no. 1 (November 2005): 529–50. http://dx.doi.org/10.1146/annurev.cellbio.21.012704.133214.
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Kean, L. S., R. S. Fuller, and J. W. Nichols. "Retrograde lipid traffic in yeast: identification of two distinct pathways for internalization of fluorescent-labeled phosphatidylcholine from the plasma membrane." Journal of Cell Biology 123, no. 6 (December 15, 1993): 1403–19. http://dx.doi.org/10.1083/jcb.123.6.1403.
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Digital, video-enhanced fluorescence microscopy and spectrofluorometry were used to follow the internalization into the yeast Saccharomyces cerevisiae of phosphatidylcholine molecules labeled on one acyl chain with the fluorescent probe 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD). Two pathways were found: (1) transport by endocytosis to the vacuole and (2) transport by a non-endocytic pathway to the nuclear envelope and mitochondria. The endocytic pathway was inhibited at low temperature (< 2 degrees C) and by ATP depletion. Mutations in secretory (SEC) genes that are necessary for membrane traffic through the secretory pathway (including SEC1, SEC2, SEC4, SEC6, SEC7, SEC12, SEC14, SEC17, SEC18, and SEC21) almost completely blocked endocytic uptake. In contrast, mutations in the SEC63, SEC65, or SEC11 genes, required for translocation of nascent secretory polypeptides into the ER or signal peptide processing in the ER, only slightly reduced endocytic uptake. Phospholipid endocytosis was also independent of the gene encoding the clathrin heavy chain, CHC1. The correlation of biochemical analysis with fluorescence microscopy indicated that the fluorescent phosphatidylcholine was degraded in the vacuole and that degradation was, at least in part, dependent on the vacuolar proteolytic cascade. The non-endocytic route functioned with a lower cellular energy charge (ATP levels 80% reduced) and was largely independent of the SEC genes. Non-endocytic transport of NBD-phosphatidylcholine to the nuclear envelope and mitochondria was inhibited by pretreatment of cells with the sulfhydryl reagents N-ethylmaleimide and p-chloromercuribenzenesulfonic acid, suggesting the existence of protein-mediated transmembrane transfer (flip-flop) of phosphatidylcholine across the yeast plasma membrane. These data establish a link between lipid movement during secretion and endocytosis in yeast and suggest that phospholipids may also gain access to intracellular organelles through non-endocytic, protein-mediated events.
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Harty, Carol, Sabine Strahl, and Karin Römisch. "O-Mannosylation Protects Mutant Alpha-Factor Precursor from Endoplasmic Reticulum-associated Degradation." Molecular Biology of the Cell 12, no. 4 (April 2001): 1093–101. http://dx.doi.org/10.1091/mbc.12.4.1093.
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Secretory proteins that fail to fold in the endoplasmic reticulum (ER) are transported back to the cytosol and degraded by proteasomes. It remains unclear how the cell distinguishes between folding intermediates and misfolded proteins. We asked whether misfolded secretory proteins are covalently modified in the ER before export. We found that a fraction of mutant alpha-factor precursor, but not the wild type, was progressively O-mannosylated in microsomes and in intact yeast cells by proteinO-mannosyl transferase 2 (Pmt2p).O-Mannosylation increased significantly in vitro under ER export conditions, i.e., in the presence of ATP and cytosol, and this required export-proficient Sec61p in the ER membrane. Deletion ofPMT2, however, did not abrogate mutant alpha-factor precursor degradation but, rather, enhanced its turnover in intact yeast cells. In vitro, O-mannosylated mutant alpha-factor precursor was stable and protease protected, and a fraction was associated with Sec61p in the ER lumen. Thus, prolonged ER residence allows modification of exposed O-mannosyl acceptor sites in misfolded proteins, which abrogates misfolded protein export from the ER at a posttargeting stage. We conclude that there is a limited window of time during which misfolded proteins can be removed from the ER before they acquire inappropriate modifications that can interfere with disposal through the Sec61 channel.
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Xu, Dan Dan, Chun Fang Hu, Xiang You, Nan Nan Lu, and Feng Guang Gao. "Akt+ IKKα/β+ Rab5+ Signalosome Mediate the Endosomal Recruitment of Sec61 and Contribute to Cross-Presentation in Bone Marrow Precursor Cells." Vaccines 8, no. 3 (September 17, 2020): 539. http://dx.doi.org/10.3390/vaccines8030539.
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Cross-presentation in dendritic cells (DC) requires the endosomal relocations of internalized antigens and the endoplasmic reticulum protein Sec61. Despite the fact that endotoxin-containing pathogen and endotoxin-free antigen have different effects on protein kinase B (Akt) and I-kappa B Kinase α/β (IKKα/β) activation, the exact roles of Akt phosphorylation, IKKα or IKKβ activation in endotoxin-containing pathogen-derived cross-presentation are poorly understood. In this study, endotoxin-free ovalbumin supplemented with endotoxin was used as a model pathogen. We investigated the effects of endotoxin-containing pathogen and endotoxin-free antigen on Akt phosphorylation, IKKα/β activation, and explored the mechanisms that the endotoxin-containing pathogen orchestrating the endosomal recruitment of Sec61 of the cross-presentation in bone marrow precursor cells (BMPC). We demonstrated that endotoxin-containing pathogen and endotoxin-free antigen efficiently induced the phosphorylation of Akt-IKKα/β and Akt-IKKα, respectively. Endotoxin-containing pathogen derived Akt+ IKKα/β+ Rab5+ signalosome, together with augmented the recruitment of Sec61 toward endosome, lead to the increased cross-presentation in BMPC. Importantly, the endosomal recruitment of Sec61 was partly mediated by the formation of Akt+ IKKα/β+ signalosome. Thus, these data suggest that Akt+ IKKα/β+ Rab5+ signalosome contribute to endotoxin-containing pathogen-induced the endosomal recruitment of Sec61 and the superior efficacy of cross-presentation in BMPC.
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Pfeffer, Stefan, and Friedrich Förster. "Sec61: A static framework for membrane-protein insertion." Channels 10, no. 3 (January 26, 2016): 167–69. http://dx.doi.org/10.1080/19336950.2015.1125737.
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Pitonzo, David, Zhongying Yang, Yoshihiro Matsumura, Arthur E. Johnson, and William R. Skach. "Sequence-specific Retention and Regulated Integration of a Nascent Membrane Protein by the Endoplasmic Reticulum Sec61 Translocon." Molecular Biology of the Cell 20, no. 2 (January 15, 2009): 685–98. http://dx.doi.org/10.1091/mbc.e08-09-0902.
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A defining feature of eukaryotic polytopic protein biogenesis involves integration, folding, and packing of hydrophobic transmembrane (TM) segments into the apolar environment of the lipid bilayer. In the endoplasmic reticulum, this process is facilitated by the Sec61 translocon. Here, we use a photocross-linking approach to examine integration intermediates derived from the ATP-binding cassette transporter cystic fibrosis transmembrane conductance regulator (CFTR) and show that the timing of translocon-mediated integration can be regulated at specific stages of synthesis. During CFTR biogenesis, the eighth TM segment exits the ribosome and enters the translocon in proximity to Sec61α. This interaction is initially weak, and TM8 spontaneously dissociates from the translocon when the nascent chain is released from the ribosome. Polypeptide extension by only a few residues, however, results in stable TM8-Sec61α photocross-links that persist after peptidyl-tRNA bond cleavage. Retention of these untethered polypeptides within the translocon requires ribosome binding and is mediated by an acidic residue, Asp924, near the center of the putative TM8 helix. Remarkably, at this stage of synthesis, nascent chain release from the translocon is also strongly inhibited by ATP depletion. These findings contrast with passive partitioning models and indicate that Sec61α can retain TMs and actively inhibit membrane integration in a sequence-specific and ATP-dependent manner.
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Silve, S., C. Volland, C. Garnier, R. Jund, M. R. Chevallier, and R. Haguenauer-Tsapis. "Membrane insertion of uracil permease, a polytopic yeast plasma membrane protein." Molecular and Cellular Biology 11, no. 2 (February 1991): 1114–24. http://dx.doi.org/10.1128/mcb.11.2.1114.
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Uracil permease is a multispanning protein of the Saccharomyces cerevisiae plasma membrane which is encoded by the FUR4 gene and produced in limited amounts. It has a long N-terminal hydrophilic segment, which is followed by 10 to 12 putative transmembrane segments, and a hydrophilic C terminus. The protein carries seven potential N-linked glycosylation sites, three of which are in its N-terminal segment. Overexpression of this permease and specific antibodies were used to show that uracil permease undergoes neither N-linked glycosylation nor proteolytic processing. Uracil permease N-terminal segments of increasing lengths were fused to a reporter glycoprotein, acid phosphatase. The in vitro and in vivo fates of the resulting hybrid proteins were analyzed to identify the first signal anchor sequence of the permease and demonstrate the cytosolic orientation of its N-terminal hydrophilic sequence. In vivo insertion of the hybrid protein bearing the first signal anchor sequence of uracil permease into the endoplasmic reticulum membrane was severely blocked in sec61 and sec62 translocation mutants.
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Silve, S., C. Volland, C. Garnier, R. Jund, M. R. Chevallier, and R. Haguenauer-Tsapis. "Membrane insertion of uracil permease, a polytopic yeast plasma membrane protein." Molecular and Cellular Biology 11, no. 2 (February 1991): 1114–24. http://dx.doi.org/10.1128/mcb.11.2.1114-1124.1991.
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Uracil permease is a multispanning protein of the Saccharomyces cerevisiae plasma membrane which is encoded by the FUR4 gene and produced in limited amounts. It has a long N-terminal hydrophilic segment, which is followed by 10 to 12 putative transmembrane segments, and a hydrophilic C terminus. The protein carries seven potential N-linked glycosylation sites, three of which are in its N-terminal segment. Overexpression of this permease and specific antibodies were used to show that uracil permease undergoes neither N-linked glycosylation nor proteolytic processing. Uracil permease N-terminal segments of increasing lengths were fused to a reporter glycoprotein, acid phosphatase. The in vitro and in vivo fates of the resulting hybrid proteins were analyzed to identify the first signal anchor sequence of the permease and demonstrate the cytosolic orientation of its N-terminal hydrophilic sequence. In vivo insertion of the hybrid protein bearing the first signal anchor sequence of uracil permease into the endoplasmic reticulum membrane was severely blocked in sec61 and sec62 translocation mutants.
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Sun, Sha, and Malaiyalam Mariappan. "C-terminal tail length guides insertion and assembly of membrane proteins." Journal of Biological Chemistry 295, no. 46 (September 2, 2020): 15498–510. http://dx.doi.org/10.1074/jbc.ra120.012992.
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A large number of newly synthesized membrane proteins in the endoplasmic reticulum (ER) are assembled into multiprotein complexes, but little is known about the mechanisms required for assembly membrane proteins. It has been suggested that membrane chaperones might exist, akin to the molecular chaperones that stabilize and direct the assembly of soluble protein complexes, but the mechanisms by which these proteins would bring together membrane protein components is unclear. Here, we have identified that the tail length of the C-terminal transmembrane domains (C-TMDs) determines efficient insertion and assembly of membrane proteins in the ER. We found that membrane proteins with C-TMD tails shorter than ∼60 amino acids are poorly inserted into the ER membrane, which suggests that translation is terminated before they are recognized by the Sec61 translocon for insertion. These C-TMDs with insufficient hydrophobicity are post-translationally recognized and retained by the Sec61 translocon complex, providing a time window for efficient assembly with TMDs from partner proteins. Retained TMDs that fail to assemble with their cognate TMDs are slowly translocated into the ER lumen and are recognized by the ER-associated degradation (ERAD) pathway for removal. In contrast, C-TMDs with sufficient hydrophobicity or tails longer than ∼80 residues are quickly released from the Sec61 translocon into the membrane or the ER lumen, resulting in inefficient assembly with partner TMDs. Thus, our data suggest that C-terminal tails harbor crucial signals for both the insertion and assembly of membrane proteins.
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Shrimal, Shiteshu, Natalia A. Cherepanova, Elisabet C. Mandon, Sergey V. Venev, and Reid Gilmore. "Asparagine-linked glycosylation is not directly coupled to protein translocation across the endoplasmic reticulum in Saccharomyces cerevisiae." Molecular Biology of the Cell 30, no. 21 (October 1, 2019): 2626–38. http://dx.doi.org/10.1091/mbc.e19-06-0330.
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Mammalian cells express two oligosaccharyltransferase complexes, STT3A and STT3B, that have distinct roles in N-linked glycosylation. The STT3A complex interacts directly with the protein translocation channel to mediate glycosylation of proteins using an N-terminal–to–C-terminal scanning mechanism. N-linked glycosylation of proteins in budding yeast has been assumed to be a cotranslational reaction. We have compared glycosylation of several glycoproteins in yeast and mammalian cells. Prosaposin, a cysteine-rich protein that contains STT3A-dependent glycosylation sites, is poorly glycosylated in yeast cells and STT3A-deficient human cells. In contrast, a protein with extreme C-terminal glycosylation sites was efficiently glycosylated in yeast by a posttranslocational mechanism. Posttranslocational glycosylation was also observed for carboxypeptidase Y–derived reporter proteins that contain closely spaced acceptor sites. A comparison of two recent protein structures indicates that the yeast OST is unable to interact with the yeast heptameric Sec complex via an evolutionarily conserved interface due to occupation of the OST binding site by the Sec63 protein. The efficiency of glycosylation in yeast is not enhanced for proteins that are translocated by the Sec61 or Ssh1 translocation channels instead of the Sec complex. We conclude that N-linked glycosylation and protein translocation are not directly coupled in yeast cells.