A posttargeting signal sequence recognition event in the endoplasmic reticulum membrane

Jungnickel, Berit; Rapoport, Tom A.

doi:10.1016/0092-8674(95)90313-5

Cited by 256 publications

(310 citation statements)

References 29 publications

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“…The membrane binding of an RNC-SRP complex could thus be mediated by both ribosome-Sec61 and SRP-SR interactions, explaining why nontranslating ribosomes would not be able to bind to the free Sec61 population. Once the nascent chain has inserted into the channel, the ribosome-Sec61 interaction is stabilized, and both SRP and SR dissociate (Jungnickel and Rapoport, 1995). When all Sec61 binding sites on the ribosome are exposed, a tetramer of Sec61 complexes could form underneath the ribosome, further stabilizing the translocating RNC at the ER membrane.…”

Section: Discussionmentioning

confidence: 98%

“…To study the membrane targeting of RNCs, mRNA coding for ppl86, lacking a stop codon, was translated in a wheat germ extract, generating stalled ribosomes with nascent chains associated as peptidyl-tRNAs (Gilmore et al, 1991;Jungnickel and Rapoport, 1995). These RNCs could bind to RMs that were stripped of ribosomes by puromycin/high salt treatment (PK-RMs; Supplemental Figure 1A).…”

Section: Testing the Membrane Dissociation Of Prebound Ribosomesmentioning

confidence: 99%

“…mRNAs coding for the N-terminal 86 amino acids of bovine preprolactin (ppl86) or for the N-terminal 120 amino acids of firefly (Photinus pyralis) luciferase (luc120) were translated for 20 min in the presence of [ 35 S]methionine in a wheat germ extract (at 28°C) or in reticulocyte lysate (at 30°C), as described previously (Jungnickel and Rapoport, 1995). The total ribosome concentration in the in vitro translation mixture was ϳ0.3 M. Translation was stopped by the addition of 2 M edeine.…”

Section: Synthesis and Isolation Of Rncsmentioning

confidence: 99%

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Ribosome Binding to and Dissociation from Translocation Sites of the Endoplasmic Reticulum Membrane

Schaletzky

Rapoport

2006

MBoC

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We have addressed how ribosome-nascent chain complexes (RNCs), associated with the signal recognition particle (SRP), can be targeted to Sec61 translocation channels of the endoplasmic reticulum (ER) membrane when all binding sites are occupied by nontranslating ribosomes. These competing ribosomes are known to be bound with high affinity to tetramers of the Sec61 complex. We found that the membrane binding of RNC-SRP complexes does not require or cause the dissociation of prebound nontranslating ribosomes, a process that is extremely slow. SRP and its receptor target RNCs to a free population of Sec61 complex, which associates with nontranslating ribosomes only weakly and is conformationally different from the population of ribosome-bound Sec61 complex. Taking into account recent structural data, we propose a model in which SRP and its receptor target RNCs to a Sec61 subpopulation of monomeric or dimeric state. This could explain how RNC-SRP complexes can overcome the competition by nontranslating ribosomes. INTRODUCTIONCotranslational translocation is the major pathway in mammals by which proteins are transported across or integrated into the endoplasmic reticulum (ER) membrane. The synthesis of these proteins starts with a free ribosome in the cytosol. Once a hydrophobic signal sequence or transmembrane (TM) segment has emerged from the ribosome, the signal recognition particle (SRP) binds, forming a ribosomenascent chain (RNC)-SRP complex (for reviews, see Walter and Johnson, 1994;Egea et al., 2005). This complex binds to the ER membrane by interactions between SRP and the SRP receptor (SR) and between the ribosome and the Sec61 complex, a heterotrimeric membrane protein that forms a proteinconducting channel (Van den Berg et al., 2004). The nascent polypeptide chain forms a loop when inserting into the channel, with the signal or TM sequence intercalated into the walls of the channel and the following mature region in the pore proper (for reviews, see Rapoport et al., 2004;Osborne et al., 2005). For secretory proteins, this part of the elongating chain is moved through the channel into the ER lumen, and the signal sequence is eventually cleaved off. In membrane proteins, TM sequences are partitioned sideways through the walls of the channel into the lipid phase, leaving some parts of the polypeptide chain in the cytosol and others in the ER lumen. For both classes of proteins, the translating ribosome stays bound to the channel during the entire translocation process (Mothes et al., 1997).Previous work has shown that the Sec61 complex can bind not only RNCs but also nontranslating ribosomes with nanomolar affinity (Borgese et al., 1974;Kalies et al., 1994;Prinz et al., 2000a). Ribosomes remain bound to ER membranes after nascent chain release (Adelman et al., 1973) and do not easily dissociate from the membrane (Borgese et al., 1973;Potter and Nicchitta, 2002). Given that the concentration of free ribosomes in a secretory cell is ϳ0.3 M, 95% of the Sec61 binding sites should be occupied (Blobel and Potter, ...

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Section: Discussionmentioning

confidence: 98%

Section: Testing the Membrane Dissociation Of Prebound Ribosomesmentioning

confidence: 99%

Section: Synthesis and Isolation Of Rncsmentioning

confidence: 99%

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Ribosome Binding to and Dissociation from Translocation Sites of the Endoplasmic Reticulum Membrane

Schaletzky

Rapoport

2006

MBoC

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“…The Sec61 channel is sealed at both ends to maintain the permeability barrier across the ER membrane (Hamman et al, 1998). Channel opening for protein import is triggered by functional signal peptides that are recognized by the Sec61 channel itself (Jungnickel and Rapoport, 1995). The signal peptide of most misfolded secretory proteins, however, is cleaved off before retrograde transport is initiated.…”

Section: Introductionmentioning

confidence: 99%

O-Mannosylation Protects Mutant Alpha-Factor Precursor from Endoplasmic Reticulum-associated Degradation

Harty

Strahl

Römisch

2001

MBoC

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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 protein O-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 of PMT2, 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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“…This heterotrimeric complex is thought to be the main constituent of the translocation channel (3,12,28). Translocation experiments using proteoliposomes with translocons reconstituted from purified elements have shown the Sec61 complex to be sufficient for the formation of a membrane channel (20). However, other components, such as the TRAM protein (15,36) or the heterotetrameric translocon-associated protein (TRAP) complex (10,13), are generally required for efficient translocation.…”

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confidence: 99%

Mutation in the Trapα/Ssr1 Gene, Encoding Translocon-Associated Protein α, Results in Outflow Tract Morphogenetic Defects

Mesbah

Camus

Babinet

et al. 2006

Molecular and Cellular Biology

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Translocon-associated protein complex (TRAP) is thought to be required for efficient protein-specific translocation across the endoplasmic reticulum membrane. We created a mutation in the Trap␣ gene that leads to the synthesis of a truncated TRAP␣ protein fused to ShBle-␤-galactosidase. Analysis of Trap␣ cDNAs reveals that among three different messenger RNAs expressed in the mouse, one of them encodes a slightly larger protein that differs in its C-terminal end. This mRNA, specific for skeletal muscle and heart, is only expressed after birth. Homozygous Trap␣ mutant pups die at birth, likely as a result of severe cardiac defects. Indeed, the septation of the proximal part of the outflow tract is absent, resulting in a double-outlet right ventricle. Studies of protein secretion in transfected embryonic fibroblasts reveal that the TRAP complex does not function properly in homozygous mutant cells and confirm, in vivo, the involvement of TRAP in substratespecific translocation. Our results provide the first in vivo demonstration that a member of the TRAP complex plays a crucial role in mammalian heart development and suggest that TRAP␣ could be involved in translocation of factors necessary for maturation of endocardial cushions.

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A posttargeting signal sequence recognition event in the endoplasmic reticulum membrane

Cited by 256 publications

References 29 publications

Ribosome Binding to and Dissociation from Translocation Sites of the Endoplasmic Reticulum Membrane

Ribosome Binding to and Dissociation from Translocation Sites of the Endoplasmic Reticulum Membrane

O-Mannosylation Protects Mutant Alpha-Factor Precursor from Endoplasmic Reticulum-associated Degradation

Mutation in the Trapα/Ssr1 Gene, Encoding Translocon-Associated Protein α, Results in Outflow Tract Morphogenetic Defects

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