A signal-anchor sequence stimulates signal recognition particle binding to ribosomes from inside the exit tunnel

Abstract

Sorting of eukaryotic membrane and secretory proteins depends on recognition of ribosome-bound nascent chain signal sequences by the signal recognition particle (SRP). The current model suggests that the SRP cycle is initiated when a signal sequence emerges from the ribosomal tunnel and binds to SRP. Then elongation is slowed until the SRP-bound ribosome-nascent chain complex (RNC) is targeted to the SRP receptor in the endoplasmic reticulum (ER) membrane. The RNC is then transferred to the translocon, SRP is released, and translation resumes. Because RNCs do not target to the translocon efficiently if nascent chains become too long, the window for SRP to identify its substrates is short. We now show that a transmembrane signal-anchor sequence (SA) significantly enhances binding of SRP to RNCs even before the SA emerges from the ribosomal tunnel. In this mode, SRP does not contact the SA directly but is in close proximity to the portion of the nascent polypeptide that has already left the ribosomal tunnel. Early recruitment of SRP provides a mechanism to expand the window for substrate identification. We suggest that the dynamics of the SRP-ribosome interaction is affected not only by the direct binding of SRP to an exposed signal sequence but also by properties of the translating ribosome that are triggered from within the tunnel.

Fig. 1.

The exposed SA of Dap2 attracts SRP and disfavors binding of other ribosome-associated protein biogenesis factors to RNCs. (A) Schematic representation of nascent Pgk1 of 120 aa, and Dap2 of 54, 60, and 120 aa. The type II membrane protein Dap2 contains a SA localized between amino acids 30 and 45. The SA is indicated in dark gray; the portion of the nascent chains covered by the ribosomal tunnel is indicated in light gray. Nascent chains contained an N-terminal FLAG tag that was used for the purification of RNCs. The positions of lysines, important for chemical cross-linking, are marked as black bars. (B) SA of Dap2 and of the mutant versions, Dap2TM, and Dap2α. (C) Occupation of Dap2-120 or Pgk1–120 RNCs with RPBs in percent of RNCs. Error bars indicate the standard error of the mean (SEM). (D) Immunoblots for the quantification of Srp54, αNAC, Ssb1/2, and Nat1 on Dap2-120 RNCs exemplifies the data in C. RNCs carrying FLAG-tagged nascent Dap2 were isolated by native immunoprecipitation using αFLAG-coated beads. Aliquots of the material recovered and purified standard proteins were analyzed by immunoblotting. Signals obtained from the same exposure of a single gel are boxed. In case of the standard, numbers above the lanes give the amount of the respective purified protein loaded to the gel in picomoles or femtomoles, respectively. The standard protein His-Rps9a was supplemented with total yeast extract to ensure quantitative recovery during the procedure. Standard curves (Fig. S2) were used to determine the amount of RNCs and attached factors pulled down via the FLAG-tagged nascent chains. The background derived from an identically treated sample containing RNCs carrying the same, but nontagged, nascent chain was subtracted. The amount of His-Rps9a in the samples was divided by the factor 0.7 corresponding to Rps9a/b deviation from the mean value of ribosomal proteins previously determined (ref. and Fig. S2).

Fig. 2.

Interaction of RPBs with nascent chains. (A) ³⁵S-labeled RNCs carrying untagged nascent chains were isolated by centrifugation through a sucrose cushion containing 120 mM potassium acetate (pH 7.4) and subsequently incubated either in the absence (TOT − BS³) or presence (TOT + BS³) of the homobifunctional cross-linker BS³. Aliquots containing 4 times as much material as the TOT + BS³ samples were subjected to immunoprecipitation (IP) under denaturing conditions with antibodies directed against Ssb1, Srp54, α/βNAC, or Nat1. Samples were run on Tris-Tricine gels and subsequently analyzed by autoradiography. (B) Interaction of SRP with RNCs after treatment with low salt or high salt. The experiment was performed as described in A with the exception that RNCs carrying FLAG-tagged nascent chains were isolated although sucrose cushions containing either 120 mM or 550 mM potassium acetate. Ssb1/2 served as a control because it no longer forms a cross-link after treatment of RNCs with 550 mM potassium acetate. Asterisks indicate the relevant cross-links.

Fig. 3.

The SA of Dap2 within the ribosomal tunnel enhances SRP association with ribosomes. (A) SRP occupancy of RNCs carrying different lengths of nascent Dap2, Dap2TM, Dap2α, or Pgk1 at low salt concentration (120 mM potassium acetate). The number of amino acids in a nascent chain, exclusive of the FLAG-tag, follows the hyphen. Error bars indicate the SEM. Analysis and quantification was as in Fig. 1D and Fig. S2. (B) Dap2TM and Dap2α have lost the ability to interact with SRP and have gained the ability to interact with Ssb1. Cross-linking with BS³ was performed as described in Fig. 2A on RNCs carrying FLAG-tagged 120-residue nascent Dap2, Dap2TM, Dap2α, and Pgk1 after isolation through sucrose cushions containing 120 mM potassium acetate. Immunoprecipitations were performed with αSrp54 or αSsb1 as indicated. Srp54-CL: cross-link between the nascent chain and Srp54; Ssb1-CL: cross-link between the nascent chain and Ssb1/2.

Fig. 4.

SRP is close to nascent chains with a Dap2 SA localized within the tunnel but requires SA exposure for salt-resistant binding. (A) SRP occupancy of RNCs carrying 60-residue Pgk1, or 60-, and 120-residue nascent Dap2 after isolation at high salt concentration (550 mM potassium acetate). Error bars indicate the SEM. Analysis and quantification was as in Fig. 1D. (B) Interaction of SRP with FLAG-tagged 54-, 60-, and 120-residue nascent Dap2, Dap2TM, or Dap2α. (C) Interaction of SRP and NAC with FLAG-tagged nascent Dap2-54 or Dap2α-54. Cross-link products are labeled with an asterisk. Cross-linking with BS³ and isolation of cross-link products was as in Fig. 2A.

Fig. 5.

A Dap2 SA inside the ribosomal tunnel is not adjacent to SRP. (A) RNCs with 54-, 60-, and 120-residue Dap2 nascent chains with a single photo probe in the middle of the SA sequence are depicted. (B) ³⁵S-methionine-labeled RNCs with εANB-Lys probes at position 39 of 120-residue length FLAG-Dap2 or FLAG-Dap2α were photolyzed (+UV). As a control, a parallel sample was not illuminated with UV light (−UV). RNCs were then isolated via the FLAG tag and analyzed by autoradiography. The cross-link between Dap2-120 and Srp54 is labeled with an asterisk. (C) ³⁵S-methionine-labeled RNCs with εANB-Lys probes at position 39 of nascent FLAG-Dap2 were photolyzed (+UV) or kept in the dark (−UV) and were then isolated via the FLAG tag. A sample corresponding to 2 times the amount of the material shown in the +UV lane was subjected to immunoprecipitation under denaturing conditions using αSrp54 coupled to protein A Sepharose beads (IP αSrp54). (D) Three-stage model of SRP binding to RNCs. Stage 1: SRP (green) binds weakly to translating ribosomes (gray). This mode of interaction is independent of the length and type of nascent chain (light blue), but SRP affinity is higher than to nontranslating ribosomes. Stage 2: Upon synthesis of a SA, the interaction with the ribosome is strengthened and SRP localizes close to the exposed portion of the nascent polypeptide. The interaction is salt sensitive, suggesting that it is mainly electrostatic in nature. Stage 3: When the SA emerges from the tunnel, SRP binds to it with high affinity. The interaction is now salt resistant, thereby suggesting that hydrophobic interactions contribute significantly. In this stage, translation is arrested and the RNC–SRP complex is targeted to SRP receptor in the ER membrane. For details and references, see Discussion.