scholarly journals Interactions between Sec Complex and Prepro-α-Factor during Posttranslational Protein Transport into the Endoplasmic Reticulum

2004 ◽  
Vol 15 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Kathrin Plath ◽  
Barrie M. Wilkinson ◽  
Colin J. Stirling ◽  
Tom A. Rapoport
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Transport ◽  
Polypeptide Chain ◽  
Signal Sequence ◽  
Cross Linking ◽  
Endoplasmic Reticulum Membrane ◽  
Transmembrane Segments ◽  
Sequence Recognition ◽  
Multiple Regions ◽  
A Site

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.

Specific transmembrane segments are selectively delayed at the ER translocon during opsin biogenesis

2008 ◽  
Vol 411 (3) ◽  
pp. 495-506 ◽  
Author(s):  
Nurzian Ismail ◽  
Samuel G. Crawshaw ◽  
Benedict C. S. Cross ◽  
Anna C. Haagsma ◽  
Stephen High
Keyword(s):  
Endoplasmic Reticulum ◽  
Polypeptide Chain ◽  
Cross Linking ◽  
Relative Location ◽  
Intrinsic Properties ◽  
Site Specific ◽  
Transmembrane Segments ◽  
Nascent Chain ◽  
Polytopic Membrane Protein ◽  
A Site

A site-specific cross-linking approach was used to study the integration of TM (transmembrane) segments 4–7 of the polytopic membrane protein, opsin, at the ER (endoplasmic reticulum). We found that although TM4 exits the ER translocon rapidly, TM segments 5, 6 and 7 are all retained at the translocon until opsin biosynthesis is terminated. Furthermore, although artificial extension of the nascent chain is not sufficient to release the C-terminal region of opsin from the translocon, substitution of the native TM segment 7 with a more hydrophobic TM segment results in its rapid lateral exit into the lipid bilayer. We conclude that the intrinsic properties of a TM segment determine the timing of its membrane integration rather than its relative location within the polypeptide chain. A pronounced and prolonged association of opsin TM5 with the translocon-associated component PAT-10 was also observed, suggesting that PAT-10 may facilitate the assembly of distinct opsin subdomains during membrane integration. The results of the present study strongly support a model in which the ER translocon co-ordinates the integration of selected TM segments in response to the specific requirements of the precursor being synthesized.

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

2000 ◽  
Vol 11 (11) ◽  
pp. 3859-3871 ◽  
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.

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

1998 ◽  
Vol 142 (2) ◽  
pp. 355-364 ◽  
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.

scholarly journals Direct probing of the interaction between the signal sequence of nascent preprolactin and the signal recognition particle by specific cross-linking.

1987 ◽  
Vol 104 (2) ◽  
pp. 201-208 ◽  
Author(s):  
M Wiedmann ◽  
T V Kurzchalia ◽  
H Bielka ◽  
T A Rapoport
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Cross Linking ◽  
Endoplasmic Reticulum Membrane ◽  
Signal Recognition ◽  
Group 4 ◽  
Lysine Residues ◽  
Nascent Chain

We have studied the interaction between the signal sequence of nascent preprolactin and the signal recognition particle (SRP) during the initial events in protein translocation across the endoplasmic reticulum membrane. A new method of affinity labeling was used, whereby lysine residues, carrying the photoreactive group 4-(3-trifluoromethyldiazirino) benzoic acid in their side chains, are incorporated into a protein by means of modified lysyl-tRNA, and cross-linking to the interacting component is induced by irradiation. SRP interacts through its Mr 54,000 polypeptide component with the signal sequences of nascent preprolactin chains containing about 70 residues, and with decreasing affinity with longer chains as well; it causes inhibition of elongation. Binding of SRP is reversible and requires the nascent chain to be bound to a functional ribosome. SRP cross-linked to the signal sequence still inhibits elongation but does not prevent it completely. We conclude that SRP does not block the exit site of the polypeptide chain on the ribosome. The SRP receptor of the endoplasmic reticulum membrane displaces the signal sequence from SRP and, even if SRP is cross-linked, releases elongation arrest.

scholarly journals Stability and flexibility of marginally hydrophobic–segment stalling at the endoplasmic reticulum translocon

2016 ◽  
Vol 27 (6) ◽  
pp. 930-940 ◽  
Author(s):  
Yuichiro Kida ◽  
Yudai Ishihara ◽  
Hidenobu Fujita ◽  
Yukiko Onishi ◽  
Masao Sakaguchi
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Lipid Phase ◽  
Endoplasmic Reticulum Membrane ◽  
Dependent Manner ◽  
Transmembrane Segment ◽  
Conducting Channel ◽  
Transmembrane Segments ◽  
Lipid Environment ◽  
Sec61 Channel

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.

scholarly journals 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.

1988 ◽  
Vol 106 (4) ◽  
pp. 1093-1104 ◽  
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.

scholarly journals Detection of Transient In Vivo Interactions between Substrate and Transporter during Protein Translocation into the Endoplasmic Reticulum

1999 ◽  
Vol 10 (2) ◽  
pp. 329-344 ◽  
Author(s):  
Martin Dünnwald ◽  
Alexander Varshavsky ◽  
Nils Johnsson
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Interactions ◽  
Polypeptide Chain ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Living Cells ◽  
C Terminus ◽  
Identical Test ◽  
Split Ubiquitin

The split-ubiquitin technique was used to detect transient protein interactions in living cells. Nub, the N-terminal half of ubiquitin (Ub), was fused to Sec62p, a component of the protein translocation machinery in the endoplasmic reticulum ofSaccharomyces cerevisiae. Cub, the C-terminal half of Ub, was fused to the C terminus of a signal sequence. The reconstitution of a quasi-native Ub structure from the two halves of Ub, and the resulting cleavage by Ub-specific proteases at the C terminus of Cub, serve as a gauge of proximity between the two test proteins linked to Nub and Cub. Using this assay, we show that Sec62p is spatially close to the signal sequence of the prepro-α-factor in vivo. This proximity is confined to the nascent polypeptide chain immediately following the signal sequence. In addition, the extent of proximity depends on the nature of the signal sequence. Cub fusions that bore the signal sequence of invertase resulted in a much lower Ub reconstitution with Nub-Sec62p than otherwise identical test proteins bearing the signal sequence of prepro-α-factor. An inactive derivative of Sec62p failed to interact with signal sequences in this assay. These in vivo findings are consistent with Sec62p being part of a signal sequence-binding complex.

scholarly journals 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.

1987 ◽  
Vol 104 (6) ◽  
pp. 1705-1714 ◽  
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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