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.

Figure 1.

Testing the dissociation of ribosomes from the membrane. (A) PK-RMs were incubated with radiolabeled ribosomes and diluted 100× with buffer. Dissociation of the ribosomes at 28°C was followed by sedimentation of the membranes at different times and measuring the radioactivity in the pellet. (B) PK-RMs were preincubated with saturating amounts of purified, radiolabeled ribosomes, and reisolated. Ribosome dissociation from these membranes was analyzed by a second sedimentation, either immediately (t = 0) or after incubation with a translation mix containing RNCs carrying unlabeled ppl86, in the absence or presence of SRP, or after incubation with buffer only. In one sample (presat.), the preincubation was done with unlabeled, instead of labeled, ribosomes. The membranes were reisolated and tested for ribosome binding by incubation with radiolabeled ribosomes and sedimentation. (C) PK-RMs were presaturated with unlabeled, nontranslating ribosomes and incubated with RNCs carrying radiolabeled ppl86 under the same conditions as in B. Insertion of the nascent chain into the channel was tested by treatment with proteinase K (PK). The asterisk indicates the nascent chain fragment protected by the ribosome alone. tRNA shows the position of nonhydrolyzed ppl86-peptidyl-tRNA. (D) PK-RMs were preincubated with an excess of translation mix containing RNCs carrying radiolabeled luc120 and reisolated (lane 4). A control was performed without membranes (lane 2). The membranes with bound luc120 were then incubated with a translation mix containing RNCs carrying either unlabeled (lanes 5–12) or labeled ppl86 (lanes 17–24), or with a translation mix lacking mRNA (mock; lanes 13–16). After sedimentation of the membranes, the samples were analyzed by SDS-PAGE and autoradiography. Controls were performed in the absence of membranes (lanes 5–8, 13, 14, and 17–20).

Figure 2.

Membranes saturated with nontranslating ribosomes contain a free Sec61 population. (A) PK-RMs were preincubated with buffer (top) or with saturating concentrations of unlabeled nontranslating ribosomes (bottom) and reisolated. The membranes were solubilized in digitonin and the extract subjected to sucrose gradient centrifugation. Fractions were collected and analyzed by immunoblotting for Sec61α and SRα. The sedimentation position of the ribosomes is indicated. (B) The ribosome-saturated membranes used in A were tested for the binding of RNCs carrying radiolabeled ppl86 in the absence or presence of SRP (lanes 4 and 6). Controls were performed without membranes (lanes 1 and 2) and with untreated PK-RMs (lanes 3 and 5). (C) Ribosome-saturated or untreated PK-RMs used in A were incubated with purified, radiolabeled ribosomes. The membranes were sedimented and the radioactivity associated with them was measured. (D) Proteoliposomes containing purified Sec61 complex alone or Sec61 complex and SR were incubated with an excess of nontranslating ribosomes. The membranes were solubilized, and the extract was subjected to sucrose gradient centrifugation.

Figure 3.

A free Sec61 population detected by antibodies in native membranes. (A) PK-RMs were presaturated with nontranslating ribosomes and reisolated. The membranes were then incubated with affinity-purified antibody against the cytosolic C terminus of Sec61α, reisolated by sedimentation, washed, and solubilized in digitonin. The extract was subjected to sucrose gradient centrifugation, and fractions were analyzed by immunoblotting for IgG heavy chain (HC) and Sec61β. (B) As in A, but using an identical amount of rabbit control IgG instead of Sec61α antibodies. (C and D) As in A and B, respectively, but with RMs. (E and F) As in A and B, respectively, but with untreated PK-RMs. The sedimentation position of the ribosomes is indicated.

Figure 4.

A free Sec61 population preferentially accessible to SRP–RNC complexes. (A) PK-RMs were preincubated with increasing concentrations of purified ribosomes (0.3, 0.6, 1.3, and 1.6 μM), reisolated, and solubilized. A detergent extract was subjected to sucrose gradient centrifugation, and fractions were analyzed by immunoblotting for Sec61α. (B) Untreated PK-RMs, RMs, or RMs preincubated with 1 or 4 μM purified ribosomes were reisolated and solubilized. The extract was separated by sucrose gradient centrifugation and analyzed by immunoblotting for Sec61α. The presence of Sec61α in heavy fractions of the PK-RM sample is due to residual membrane-bound ribosomes in this particular PK-RM preparation. (C) Untreated PK-RMs or RMs were incubated in the absence of SRP with purified, radiolabeled ribosomes or with purified RNCs carrying radiolabeled ppl86. (D) The membranes used in B were incubated with RNCs carrying ppl86 in reticulocyte lysate containing SRP. Samples were placed on ice at different times and treated with proteinase K to test insertion of ppl86 into the channel. The asterisk indicates the nascent chain fragment protected by the ribosome alone. (E) Quantitation of two experiments as in D. The percentage of protected ppl86 is given.

Figure 5.

Free and ribosome-bound Sec61 populations are interconvertible. (A) RMs were solubilized in deoxy-BIGCHAP, and the detergent extract was separated by sucrose gradient centrifugation (also using deoxy-BIGCHAP). Fractions were analyzed by immunoblotting for Sec61α and SRα. (B) The fractions containing free Sec61 complex in A were pooled and reconstituted into proteoliposomes overnight. These proteoliposomes were incubated with an excess of nontranslating ribosomes and solubilized. The extract was separated by sucrose gradient centrifugation as described above, and fractions were analyzed for Sec61α and SRα. (C) The ribosome-bound fractions of Sec61 in A were reconstituted and analyzed as in B without addition of ribosomes. (D) PK-RMs were preincubated with purified, radiolabeled ribosomes and reisolated. The membranes were incubated with either buffer or 50 μg/ml chymotrypsin on ice. Proteolysis was stopped at different times, and the membranes were sedimented. The radioactivity in both supernatant and pellet was measured. (E) PK-RMs were preincubated with purified, radiolabeled ribosomes and reisolated. The membranes were incubated with buffer or 100 μM ATA at 0 or 28°C, as indicated. The dissociation of labeled ribosomes was determined by sedimentation of the membranes at different times.

Figure 6.

Sec61β–Sec61β cross-links formed in ribosome-bound Sec61 are largely reduced in free Sec61. (A) RMs were treated with 1 mM BMH for 30 min on ice. The reaction was stopped, and the membranes were solubilized. The extract was separated on a sucrose gradient and analyzed by immunoblotting for Sec61α and Sec61β. The cross-links between Sec61α and Sec61β and between Sec61β and Sec61β are indicated (α/β and β/β, respectively). (B) As in A, but in the absence of BMH. (C) Quantitation of the experiment in A. The intensities of the bands corresponding to Sec61α, the α/β cross-link (α/β), and the β/β cross-link (β/β) in both the free (black bars) and ribosome-bound (gray bars) fractions were determined by densitometry.

Figure 7.

Model for the role of SRP in targeting ribosomes to translocation sites. ER membranes contain a pool of tetrameric Sec61 complex (indicated by two neighboring gray ovals) that has a high affinity for ribosomes and is interconvertible with a pool of free Sec61 that has a low affinity for ribosomes or RNCs (gray oval) and may consist of Sec61 monomers or dimers. Ribosomes carrying a nascent chain with a signal or transmembrane sequence can interact with SRP. These complexes can bind to the SR and subsequently to free Sec61. The nascent chain is inserted into the Sec61 channel and stabilizes the complex. At a later stage of translocation, a tetramer of Sec61 complexes may form underneath the ribosome.