Signal Sequence Recognition and Protein Targeting to the Endoplasmic Reticulum Membrane

Posttranslational translocation of prepro-α-factor (ppαF) across the yeast endoplasmic reticulum membrane begins with the binding of the signal sequence to the Sec complex, a membrane component consisting of the trimeric Sec61p complex and the tetrameric Sec62p/63p complex. We show by photo-cross-linking that the signal sequence is bound directly to a site where it contacts simultaneously Sec61p and Sec62p, suggesting that there is a single signal sequence recognition step. We found no evidence for the simultaneous contact of the signal sequence with two Sec61p molecules. To identify transmembrane segments of Sec61p that line the actual translocation pore, a late translocation intermediate of ppαF was generated with photoreactive probes incorporated into the mature portion of the polypeptide. Cross-linking to multiple regions of Sec61p was observed. In contrast to the signal sequence, neighboring positions of the mature portion of ppαF had similar interactions with Sec61p. These data suggest that the channel pore is lined by several transmembrane segments, which have no significant affinity for the translocating polypeptide chain.

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Sec62p, A Component of the Endoplasmic Reticulum Protein Translocation Machinery, Contains Multiple Binding Sites for the Sec-Complex

Molecular Biology of the Cell ◽

10.1091/mbc.11.11.3859 ◽

2000 ◽

Vol 11 (11) ◽

pp. 3859-3871 ◽

Cited By ~ 32

Author(s):

Sandra Wittke ◽

Martin Dünnwald ◽

Nils Johnsson

Keyword(s):

Endoplasmic Reticulum ◽

Protein Translocation ◽

Signal Sequence ◽

Endoplasmic Reticulum Membrane ◽

Sequence Recognition ◽

Multiple Binding Sites ◽

Oligomeric Protein ◽

Multiple Binding ◽

Endoplasmic Reticulum Protein

SEC62 encodes an essential component of the Sec-complex that is responsible for posttranslational protein translocation across the membrane of the endoplasmic reticulum in Saccharomyces cerevisiae. The specific role of Sec62p in translocation was not known and difficult to identify because it is part of an oligomeric protein complex in the endoplasmic reticulum membrane. An in vivo competition assay allowed us to characterize and dissect physical and functional interactions between Sec62p and components of the Sec-complex. We could show that Sec62p binds via its cytosolic N- and C-terminal domains to the Sec-complex. The N-terminal domain, which harbors the major interaction site, binds directly to the last 14 residues of Sec63p. The C-terminal binding site of Sec62p is less important for complex stability, but adjoins the region in Sec62p that might be involved in signal sequence recognition.

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Signal Sequence Recognition in Cotranslational Translocation by Protein Components of the Endoplasmic Reticulum Membrane

The Journal of Cell Biology ◽

10.1083/jcb.142.2.355 ◽

1998 ◽

Vol 142 (2) ◽

pp. 355-364 ◽

Cited By ~ 46

Author(s):

Walther Mothes ◽

Berit Jungnickel ◽

Josef Brunner ◽

Tom A. Rapoport

Keyword(s):

Endoplasmic Reticulum ◽

Binding Site ◽

Signal Sequence ◽

Specific Binding ◽

Secretory Proteins ◽

Endoplasmic Reticulum Membrane ◽

Detergent Solution ◽

Signal Sequences ◽

Sequence Recognition ◽

Protein Components

We have investigated the role of membrane proteins and lipids during early phases of the cotranslational insertion of secretory proteins into the translocation channel of the endoplasmic reticulum (ER) membrane. We demonstrate that all steps, including the one during which signal sequence recognition occurs, can be reproduced with purified translocation components in detergent solution, in the absence of bulk lipids or a bilayer. Photocross-linking experiments with native membranes show that upon complete insertion into the channel signal sequences are both precisely positioned with respect to the protein components of the channel and contact lipids. Together, these results indicate that signal sequences are bound to a specific binding site at the interface between the channel and the surrounding lipids, and are recognized ultimately by protein–protein interactions. Our data also suggest that at least some signal sequences reach the binding site by transfer through the interior of the channel.

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The beta subunit of the signal recognition particle receptor is a transmembrane GTPase that anchors the alpha subunit, a peripheral membrane GTPase, to the endoplasmic reticulum membrane.

The Journal of Cell Biology ◽

10.1083/jcb.128.3.273 ◽

1995 ◽

Vol 128 (3) ◽

pp. 273-282 ◽

Cited By ~ 76

Author(s):

J D Miller ◽

S Tajima ◽

L Lauffer ◽

P Walter

Keyword(s):

Endoplasmic Reticulum ◽

Protein Targeting ◽

Signal Sequence ◽

Transmembrane Protein ◽

Signal Recognition Particle ◽

Endoplasmic Reticulum Membrane ◽

Protein Chain ◽

Signal Recognition ◽

Signal Recognition Particle Receptor ◽

Er Membrane

The signal recognition particle receptor (SR) is required for the cotranslational targeting of both secretory and membrane proteins to the endoplasmic reticulum (ER) membrane. During targeting, the SR interacts with the signal recognition particle (SRP) which is bound to the signal sequence of the nascent protein chain. This interaction catalyzes the GTP-dependent transfer of the nascent chain from SRP to the protein translocation apparatus in the ER membrane. The SR is a heterodimeric protein comprised of a 69-kD subunit (SR alpha) and a 30-kD subunit (SR beta) which are associated with the ER membrane in an unknown manner. SR alpha and the 54-kD subunits of SRP (SRP54) each contain related GTPase domains which are required for SR and SRP function. Molecular cloning and sequencing of a cDNA encoding SR beta revealed that SR beta is a transmembrane protein and, like SR alpha and SRP54, is a member of the GTPase superfamily. Although SR beta defines its own GTPase subfamily, it is distantly related to ARF and Sar1. Using UV cross-linking, we confirm that SR beta binds GTP specifically. Proteolytic digestion experiments show that SR alpha is required for the interaction of SRP with SR. SR alpha appears to be peripherally associated with the ER membrane, and we suggest that SR beta, as an integral membrane protein, mediates the membrane association of SR alpha. The discovery of its guanine nucleotide-binding domain, however, makes it likely that its role is more complex than that of a passive anchor for SR alpha. These findings suggest that a cascade of three directly interacting GTPases functions during protein targeting to the ER membrane.

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A posttargeting signal sequence recognition event in the endoplasmic reticulum membrane

Cell ◽

10.1016/0092-8674(95)90313-5 ◽

1995 ◽

Vol 82 (2) ◽

pp. 261-270 ◽

Cited By ~ 184

Author(s):

Berit Jungnickel ◽

Tom A Rapoport

Keyword(s):

Endoplasmic Reticulum ◽

Signal Sequence ◽

Endoplasmic Reticulum Membrane ◽

Sequence Recognition

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Alternative redox forms of ASNA-1 separate insulin signaling from tail-anchored protein targeting and cisplatin resistance in C. elegans

Scientific Reports ◽

10.1038/s41598-021-88085-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Dorota Raj ◽

Ola Billing ◽

Agnieszka Podraza-Farhanieh ◽

Bashar Kraish ◽

Oskar Hemmingsson ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Insulin Secretion ◽

Protein Targeting ◽

Cisplatin Resistance ◽

Acquired Resistance ◽

Endoplasmic Reticulum Membrane ◽

C Elegans ◽

Cisplatin Sensitivity ◽

Tumor Environment ◽

The One

AbstractCisplatin is a frontline cancer therapeutic, but intrinsic or acquired resistance is common. We previously showed that cisplatin sensitivity can be achieved by inactivation of ASNA-1/TRC40 in mammalian cancer cells and in Caenorhabditis elegans. ASNA-1 has two more conserved functions: in promoting tail-anchored protein (TAP) targeting to the endoplasmic reticulum membrane and in promoting insulin secretion. However, the relation between its different functions has remained unknown. Here, we show that ASNA-1 exists in two redox states that promote TAP-targeting and insulin secretion separately. The reduced state is the one required for cisplatin resistance: an ASNA-1 point mutant, in which the protein preferentially was found in the oxidized state, was sensitive to cisplatin and defective for TAP targeting but had no insulin secretion defect. The same was true for mutants in wrb-1, which we identify as the C. elegans homolog of WRB, the ASNA1/TRC40 receptor. Finally, we uncover a previously unknown action of cisplatin induced reactive oxygen species: cisplatin induced ROS drives ASNA-1 into the oxidized form, and selectively prevents an ASNA-1-dependent TAP substrate from reaching the endoplasmic reticulum. Our work suggests that ASNA-1 acts as a redox-sensitive target for cisplatin cytotoxicity and that cisplatin resistance is likely mediated by ASNA-1-dependent TAP substrates. Treatments that promote an oxidizing tumor environment should be explored as possible means to combat cisplatin resistance.

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Targeting of the hepatitis B virus precore protein to the endoplasmic reticulum membrane: after signal peptide cleavage translocation can be aborted and the product released into the cytoplasm.

The Journal of Cell Biology ◽

10.1083/jcb.106.4.1093 ◽

1988 ◽

Vol 106 (4) ◽

pp. 1093-1104 ◽

Cited By ~ 126

Author(s):

P D Garcia ◽

J H Ou ◽

W J Rutter ◽

P Walter

Keyword(s):

Endoplasmic Reticulum ◽

Protein Translocation ◽

Core Protein ◽

Signal Sequence ◽

Signal Peptidase ◽

Amino Terminus ◽

Endoplasmic Reticulum Membrane ◽

Nucleic Acid Binding ◽

Precore Region ◽

B Virus

The major hepatitis B virus (HBV) core protein is a viral structural protein involved in nucleic acid binding. Its coding sequence contains an extension of 29 codons (the "precore" region) at the amino terminus of the protein which is present in a fraction of the viral transcripts. This region is evolutionarily conserved among mammalian and avian HBVs, suggesting it has functional importance, although at least for duck HBV it has been shown to be nonessential for replication of infectious virions. Using in vitro assays for protein translocation across the endoplasmic reticulum membrane, we found that the precore region of the HBV genome encodes a signal sequence. This signal sequence was recognized by signal recognition particle, which targeted the nascent precore protein to the endoplasmic reticulum membrane with efficiencies comparable to those of other mammalian secretory proteins. A 19-amino acid signal peptide was removed by signal peptidase on the lumenal side of the microsomal membrane, generating a protein similar to the HBV major core protein, but containing 10 additional amino acids from the precore region at its amino terminus. Surprisingly, we found that 70-80% of this signal peptidase-cleaved product was localized on the cytoplasmic side of the microsomal vesicles and was not associated with the membranes. We conclude that translocation was aborted by an unknown mechanism, then the protein disengaged from the translocation machinery and was released back into the cytoplasm. Thus, a cytoplasmically disposed protein was created whose amino terminus resulted from signal peptidase cleavage. The remaining 20-30% appeared to be completely translocated into the lumen of the microsomes. A deletion mutant lacking the carboxy-terminal nucleic acid binding domain of the precore protein was similarly partitioned between the lumen of the microsomes and the cytoplasmic compartment, indicating that this highly charged domain is not responsible for the aborted translocation. We discuss the implications of our findings for the protein translocation process and suggest a possible role in the virus life cycle.

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The influenza hemagglutinin insertion signal is not cleaved and does not halt translocation when presented to the endoplasmic reticulum membrane as part of a translocating polypeptide.

The Journal of Cell Biology ◽

10.1083/jcb.104.6.1705 ◽

1987 ◽

Vol 104 (6) ◽

pp. 1705-1714 ◽

Cited By ~ 19

Author(s):

J Finidori ◽

L Rizzolo ◽

A Gonzalez ◽

G Kreibich ◽

M Adesnik ◽

...

Keyword(s):

Amino Acids ◽

Endoplasmic Reticulum ◽

Signal Sequence ◽

In Vitro Translation ◽

Signal Peptidase ◽

Endoplasmic Reticulum Membrane ◽

Hydrophobic Region ◽

Amino Terminal ◽

Influenza Hemagglutinin

The co-translational insertion of polypeptides into endoplasmic reticulum membranes may be initiated by cleavable amino-terminal insertion signals, as well as by permanent insertion signals located at the amino-terminus or in the interior of a polypeptide. To determine whether the location of an insertion signal within a polypeptide affects its function, possibly by affecting its capacity to achieve a loop disposition during its insertion into the membrane, we have investigated the functional properties of relocated insertion signals within chimeric polypeptides. An artificial gene encoding a polypeptide (THA-HA), consisting of the luminal domain of the influenza hemagglutinin preceded by its amino-terminal signal sequence and linked at its carboxy-terminus to an intact prehemagglutinin polypeptide, was constructed and expressed in in vitro translation systems containing microsomal membranes. As expected, the amino-terminal signal initiated co-translational insertion of the hybrid polypeptide into the membranes. The second, identical, interiorized signal, however, was not recognized by the signal peptidase and was translocated across the membrane. The failure of the interiorized signal to be cleaved may be attributed to the fact that it enters the membrane as part of a translocating polypeptide and therefore cannot achieve the loop configuration that is thought to be adopted by signals that initiate insertion. The finding that the interiorized signal did not halt translocation of downstream sequences, even though it contains a hydrophobic region and must enter the membrane in the same configuration as natural stop-transfer signals, indicates that the HA insertion signal lacks essential elements of halt transfer signals that makes the latter effective membrane-anchoring domains. When the amino-terminal insertion signal of the THA-HA chimera was deleted, the interior signal was incapable of mediating insertion, probably because of steric hindrance by the folded preceding portions of the chimera. Several chimeras were constructed in which the interiorized signal was preceded by polypeptide segments of various lengths. A signal preceded by a segment of 111 amino acids was also incapable of initiating insertion, but insertion took place normally when the segment preceding the signal was only 11-amino acids long.(ABSTRACT TRUNCATED AT 400 WORDS)

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Structural and functional characterization of Sec66p, a new subunit of the polypeptide translocation apparatus in the yeast endoplasmic reticulum.

Molecular Biology of the Cell ◽

10.1091/mbc.4.9.931 ◽

1993 ◽

Vol 4 (9) ◽

pp. 931-939 ◽

Cited By ~ 52

Author(s):

D Feldheim ◽

K Yoshimura ◽

A Admon ◽

R Schekman

Keyword(s):

Endoplasmic Reticulum ◽

Signal Sequence ◽

Functional Characterization ◽

Yeast Cells ◽

Temperature Sensitive ◽

Endoplasmic Reticulum Membrane ◽

Restrictive Temperature ◽

Permissive Temperature

SEC66 encodes the 31.5-kDa glycoprotein of the Sec63p complex, an integral endoplasmic reticulum membrane protein complex required for translocation of presecretory proteins in Saccharomyces cerevisiae. DNA sequence analysis of SEC66 predicts a 23-kDa protein with no obvious NH2-terminal signal sequence but with one domain of sufficient length and hydrophobicity to span a lipid bilayer. Antibodies directed against a recombinant form of Sec66p were used to confirm the membrane location of Sec66p and that Sec66p is a glycoprotein of 31.5 kDa. A null mutation in SEC66 renders yeast cells temperature sensitive for growth. sec66 cells accumulate some secretory precursors at a permissive temperature and a variety of precursors at the restrictive temperature. sec66 cells show defects in Sec63p complex formation. Because sec66 cells affect the translocation of some, but not all secretory precursor polypeptides, the role of Sec66p may be to interact with the signal peptide of presecretory proteins.

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NAC functions as a modulator of SRP during the early steps of protein targeting to the endoplasmic reticulum

Molecular Biology of the Cell ◽

10.1091/mbc.e12-02-0112 ◽

2012 ◽

Vol 23 (16) ◽

pp. 3027-3040 ◽

Cited By ~ 41

Author(s):

Ying Zhang ◽

Uta Berndt ◽

Hanna Gölz ◽

Arlette Tais ◽

Stefan Oellerer ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Protein Targeting ◽

Signal Sequence ◽

Signal Recognition Particle ◽

Signal Recognition ◽

Signal Sequences ◽

Chain Complexes ◽

Ribosomal Tunnel ◽

Nascent Chain ◽

Combined Data

Nascent polypeptide-associated complex (NAC) was initially found to bind to any segment of the nascent chain except signal sequences. In this way, NAC is believed to prevent mistargeting due to binding of signal recognition particle (SRP) to signalless ribosome nascent chain complexes (RNCs). Here we revisit the interplay between NAC and SRP. NAC does not affect SRP function with respect to signalless RNCs; however, NAC does affect SRP function with respect to RNCs targeted to the endoplasmic reticulum (ER). First, early recruitment of SRP to RNCs containing a signal sequence within the ribosomal tunnel is NAC dependent. Second, NAC is able to directly and tightly bind to nascent signal sequences. Third, SRP initially displaces NAC from RNCs; however, when the signal sequence emerges further, trimeric NAC·RNC·SRP complexes form. Fourth, upon docking to the ER membrane NAC remains bound to RNCs, allowing NAC to shield cytosolically exposed nascent chain domains not only before but also during cotranslational translocation. The combined data indicate a functional interplay between NAC and SRP on ER-targeted RNCs, which is based on the ability of the two complexes to bind simultaneously to distinct segments of a single nascent chain.

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