Signal Recognition Particle-dependent Targeting of Ribosomes to the Rough Endoplasmic Reticulum in the Absence and Presence of the Nascent Polypeptide-associated Complex

Raden, David; Gilmore, Reid

doi:10.1091/mbc.9.1.117

Cited by 50 publications

(59 citation statements)

References 45 publications

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“…These RNCs could bind to RMs that were stripped of ribosomes by puromycin/high salt treatment (PK-RMs; Supplemental Figure 1A). As reported previously, the preincubation of PK-RMs with an excess of nontranslating ribosomes significantly reduced RNC binding, unless SRP was present (Supplemental Figure 1A; Neuhof et al, 1998;Raden and Gilmore, 1998). Similar results were obtained with proteoliposomes containing purified Sec61 complex and SR (Supplemental Figure 1B; Neuhof et al, 1998).…”

Section: Testing the Membrane Dissociation Of Prebound Ribosomessupporting

confidence: 84%

“…The occupation of ribosome binding sites raises the question of how ribosomes that carry nascent chains destined for translocation can efficiently be targeted to Sec61. Experiments in vitro have shown that SRP is required for the binding of RNCs in the presence, but not absence, of competing ribosomes (Neuhof et al, 1998;Raden and Gilmore, 1998). SRP promotes RNC targeting both when competing ribosomes are present during the binding reaction and when they are prebound to the membrane.…”

Section: Introductionmentioning

confidence: 99%

“…SRP promotes RNC targeting both when competing ribosomes are present during the binding reaction and when they are prebound to the membrane. Together, these results suggest that the major role of SRP may be to provide RNC complexes a selective advantage in membrane targeting so that they can overcome the competition by ribosomes not engaged in translocation (Neuhof et al, 1998;Raden and Gilmore, 1998). However, how exactly SRP binding facilitates RNC targeting to Sec61 is unclear.…”

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confidence: 98%

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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: Testing the Membrane Dissociation Of Prebound Ribosomessupporting

confidence: 84%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 98%

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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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“…Alternatively NAC might modulate the interaction of SRP with the ribosome (Powers and Walter, 1996) or the interaction of RNCs with the ER (Lauring et al, 1995a,b;Mö ller et al, 1998). The latter finding has been challenged (Neuhof et al, 1998;Raden and Gilmore, 1998). Recent experiments describe yeast NAC as a ribosome-associated complex involved in mitochondrial protein import and possibly targeting to the ER (George et al, 1998).…”

Section: Purification Of Nac As An Import Stimulatory Factormentioning

confidence: 99%

Nascent Polypeptide–associated Complex Stimulates Protein Import into Yeast Mitochondria

Fünfschilling

Rospert

1999

MBoC

148

127

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To identify yeast cytosolic proteins that mediate targeting of precursor proteins to mitochondria, we developed an in vitro import system consisting of purified yeast mitochondria and a radiolabeled mitochondrial precursor protein whose C terminus was still attached to the ribosome. In this system, the N terminus of the nascent chain was translocated across both mitochondrial membranes, generating a translocation intermediate spanning both membranes. The nascent chain could then be completely chased into the mitochondrial matrix after release from the ribosome. Generation of this import intermediate was dependent on a mitochondrial membrane potential, mitochondrial surface proteins, and was stimulated by proteins that could be released from the ribosomes by high salt. The major salt-released stimulatory factor was yeast nascent polypeptide–associated complex (NAC). Purified NAC fully restored import of salt-washed ribosome-bound nascent chains by enhancing productive binding of the chains to mitochondria. We propose that ribosome-associated NAC facilitates recognition of nascent precursor chains by the mitochondrial import machinery.

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“…Adding back purified NAC to the system restored the fidelity of cotranslational ribosome binding and translocation of only secretory polypeptides (10). Recently the function of NAC in ribosome targeting to the ER was questioned (11,12) in studies that used subphysiological concentrations of NAC. Although we were able to reproduce these results, we found that, when NAC was restored to physiological levels, our original observations were confirmed (1).…”

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confidence: 99%

The α and β Subunit of the Nascent Polypeptide-associated Complex Have Distinct Functions

Beatrix

Sakai

Wiedmann

2000

Journal of Biological Chemistry

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Nascent polypeptide-associated complex (NAC) is probably the first cytosolic protein to contact nascent polypeptide chains emerging from ribosomes. In this way NAC prevents inappropriate interactions with other factors. Eventually other factors involved in targeting and folding, like the Signal Recognition Particle or cytosolic chaperones, must gain access to the nascent chain. All NAC preparations to date consist of two copurifying polypeptides. Here we rigorously show that these two polypeptides, termed ␣-and ␤NAC, form a very stable complex in vivo and in vitro and that a functional complex can be reconstituted from the individual subunits. A dissection of the contributions of the individual subunits to NACs function revealed that both subunits are in direct contact with nascent polypeptide chains on the ribosome and that both contribute to the prevention of inappropriate interactions. However, ␤NAC alone directly binds to the ribosome and is sufficient to prevent ribosome binding to the endoplasmic reticulum membrane. Nascent polypeptide-associated complex (NAC) 1 is a very abundant cytosolic protein, which is involved in cotranslational targeting of polypeptides to the endoplasmic reticulum (ER) membrane. The intracellular NAC concentration varies only slightly in different tissues, ranging from 3 to 10 M (1). NAC is highly conserved among eukaryotes, but up to now no functional prokaryotic homolog has been described. The importance of NACs in vivo function is emphasized by early embryonically lethal phenotypes of NAC mutants in mice and fruit flies (2, 3). Recently it was also shown that yeast NAC, although not essential for growth (4), is involved in the import of proteins into mitochondria (5, 6).NAC was originally identified as a ribosome-associated protein when we set out to probe the molecular environment of growing polypeptides on the ribosome using a photo-cross-linking approach (7). With this technique we were able to show that NAC can interact with regions of nascent chains as close as 17 amino acids to the peptidyl transferase center. Additionally, it was shown by protease protection that NAC functions as a dissociable wall of a ribosomal tunnel through which the growing polypeptide emerges (8). Cycles of binding and releasing NAC expose the polypeptide to the cytosol in "quantal units" rather than amino acid by amino acid. We have proposed that exposing the polypeptide chain in functional units contributes to fidelity in cotranslational processes such as targeting and folding (9). Depleting NAC from translating ribosomes led to at least two inappropriate interactions. First, the signal recognition particle (SRP), which interacts with signal peptides of nascent chains on ribosomes during targeting to the ER membrane, could be cross-linked to nascent chains lacking signal peptides (7). Second, in the absence of NAC ribosomes translating non-secretory polypeptides inappropriately interacted with translocation sites on ER membranes and the signal-less chains were even translocated, although with l...

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Signal Recognition Particle-dependent Targeting of Ribosomes to the Rough Endoplasmic Reticulum in the Absence and Presence of the Nascent Polypeptide-associated Complex

Cited by 50 publications

References 45 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

Nascent Polypeptide–associated Complex Stimulates Protein Import into Yeast Mitochondria

The α and β Subunit of the Nascent Polypeptide-associated Complex Have Distinct Functions

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