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

Abstract

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, ...

Visualization of Distinct Entities of the SecYEG Translocon during Translocation and Integration of Bacterial Proteins

“…By recruiting a YidC dimer the 500-kDa complex would be converted into a 640-kDa complex, which would be in line with the complex running at ϳ600 kDa on BN-PAGE ( Figure 5). This speculative assembly scheme would be similar to the proposed assembly of the eukaryotic Sec61 complex, which has been shown to assemble upon ribosome contact as a dimer of dimers, to which a dimer of TRAP, the translocon-associated protein, is bound (Menetret et al, 2005;Schaletzky and Rapoport, 2006). On the other hand, it is important to emphasize that the detection of complexes by BN-PAGE primarily depicts their stability and not necessarily the order of their formation.…”

Regulation of the Protein-Conducting Channel by a Bound Ribosome

“…The ribosome-lipid connection allows the ribosome to maintain its angle relative to the membrane plane, estimated to be 20° based on the cryo-EM density and, thus, to maintain also the gap between ribosome and channel (Ménétret et al, 2007). The ribosome-membrane contact is a feature of the overall placement of SecY as seen in the EM map and should not be affected by long-time relaxation; however, the contact may be superseded by interactions with a second copy of SecY, as experiments have indicated additional copies of the channel may be present in functioning ribosome-translocon complexes (Schaletzky and Rapoport, 2006). …”

Constitutive, translation-independent opening of the protein-conducting channel in the endoplasmic reticulum

“…All of the dose-response data were fitted well by a single, highaffinity component, without any evidence at the highest concentrations tested (Fig. 2a) that the entry of 4MαG was also produced by the binding of 60S subunits to a lowaffinity binding site identified as monomers of Sec61α [38]. By performing these assays at a physiological salt concentration (140 mM potassium glutamate), the binding of 60S subunits to receptors other than the Sec61 complex should have been prevented [19].…”