Signal recognition particle mediates post-translational targeting in eukaryotes

Abstract

Signal recognition particle (SRP) plays a central role in the delivery of classical secretory and membrane proteins to the endoplasmic reticulum (ER). All nascent chains studied to date dissociate from SRP once released from the ribosome, thereby supporting a strictly cotranslational mode of action for eukaryotic SRP. We now report a novel post-translational function for SRP in the targeting of tail-anchored (TA) proteins to the ER. TA proteins possess a hydrophobic membrane insertion sequence at their C-terminus such that it can only emerge from the ribosome after translation is terminated. We show that SRP can associate post-translationally with this type of ER-targeting signal, and deliver newly synthesised TA proteins to the ER membrane by a pathway dependent upon GTP and the SRP receptor. We find that dependency upon this SRP-dependent route is precursor specific, and propose a unifying model to describe the biogenesis of TA proteins in vivo.

Figure 1

TA proteins associate with SRP54 post-translationally. (A) Stop codon minus RNA encoding full-length Syb2 (lanes 5–9), full-length Sec61β (lanes 10–14) or the first 86 residues of preprolactin (lanes 1–4) was translated for 20 min, and synthesis then terminated by the addition of cycloheximide (CHX) or puromycin (puro) as shown. Crosslinking was induced with DSS as indicated. Total products were analysed directly (lanes 1, 2, 5, 6, 10 and 11) or following immunoprecipitation with antisera for SRP54 (αSRP54), ubiquitin (αUb) or a nonrelated serum (NRS). (B) Amino-acid sequences of the hydrophobic ER-targeting signals from the polypeptides used in this study. Potential TM domains are underlined, while dots indicate regions where hydrophilic domains of the polypeptide extend beyond the sequence presented. Numbers in superscript show the total length of the various polypeptides used. Where two numbers are given, the first is the limit of the sequence presented and the second the length of the polypeptide studied. (C) Cell-free translations of the various precursors were terminated with puromycin and the samples treated with DSS. In order to avoid any variability resulting from differences in translation efficiency, a fraction of each sample was first analysed by SDS–PAGE and the relative amount of each nascent chain determined by quantitative phosphorimaging (data not shown). On the basis of this analysis, equivalent amounts of each DSS-treated radiolabelled precursor were then used for the immunoprecipitation of adducts with SRP54. (D) Stop codon minus RNA was translated for 20 min in the presence or absence of canine pancreatic microsomes, and then incubated with puromycin for a further 20 min to enable membrane integration. The membrane fraction was isolated by centrifugation through a high-salt sucrose cushion. The resulting pellet was then resuspended in alkaline sodium carbonate solution and the membrane pellet re-isolated by centrifugation. For each precursor, the translation reaction lacking any exogenously added membranes was processed in parallel to provide an estimate of the amount of each protein that was recovered in the final pellet fraction independent of membrane integration. The amount of precursor present in each of the fractions was determined by SDS–PAGE and quantitative phosphorimaging, and was expressed as a percentage of the total protein synthesised in that reaction. The final value for membrane integration is the percentage of the total protein synthesised, which was specifically recovered with the isolated microsomal membranes (see Supplementary data). These values are the means of two or more independent experiments, and standard errors are indicated.

Figure 2

TA protein association with SRP54 is strictly post-translational. Syb2 or PPL86 RNA was translated for 5 min, treated with cycloheximide or puromycin, and crosslinked using DSS. RNCs were pelleted through a high-salt cushion (pel), leaving free polypeptides in the supernatant (sup). A fraction of each sample was analysed prior to crosslinking (PPL85 and Syb2), while the remainder was used for the immunoprecipitation of adducts with NACα and SRP54 as indicated.

Figure 3

SRP associates with the hydrophobic domain of TA proteins. Stop codon minus RNAs encoding the indicated polypeptides with either a single cysteine probe (upper panel) or a leucine at the equivalent location (lower panel) was translated for 15 min. The resulting RNCs were then isolated through a low-salt cushion to separate the precursors from DTT in the translation reaction and facilitate crosslinking with the bifunctional maleimide o-PDM. Nascent chains were released from ribosomes with puromycin, treated with DSS or o-PDM, and adducts with SRP54 recovered by immunoprecipitation. The relative efficiency with which each precursor was synthesised was established by quantitative phophorimaging prior to immunoprecipitation, and equivalent amounts of radiolabelled polypeptides were used for subsequent immunoisolation as for Figure 1C.

Figure 4

Membrane insertion of Syb2 is GTP dependent. (A) Flow diagram of the assay used to investigate the nucleotide dependence of Syb2 integration following apyrase treatment. (B) Syb2 RNA was translated for 20 min and treated with puromycin. Samples were depleted of nucleotide di- and triphosphates by treatment with apyrase, or mock treated. Aliquots were supplemented with combinations of AMPPNP, GMPPNP and RMs. After a 5 min incubation, the extent of membrane integration was assessed using sodium carbonate extraction, and SRP release assessed by DSS crosslinking and immunoprecipitation with αSRP54. Relative membrane integration (see the upper panel) was calculated by quantifying the increase in sodium carbonate-resistant material upon the addition of RMs (+), relative to a control sample with no added RMs (−), cf. Supplementary data. Mock-treated membranes were set at 100% relative membrane integration (lane 2). SRP release (lower panel) indicates the reduction in the amount of SRP54 adduct caused by the addition of RMs.

Figure 5

Membrane insertion of Syb2 is dependent on the SRP receptor. (A) Syb2 RNA lacking a stop codon was translated for 20 min and then treated with puromycin. RMs were treated with trypsin or mock treated as indicated, re-isolated by centrifugation, and added to the translation mix with or without soluble SR. After a 5 min incubation, membrane integration was assessed by resistance to sodium carbonate extraction. Samples were separated by SDS–PAGE. Quantification of membrane integration was performed as described for Figure 4B, with mock-treated RM set to 100%. Lane 1 shows the level of background signal obtained in the absence of any added membranes. This contribution was subtracted from the signals obtained in the presence of membranes during quantification, and is therefore labelled as 0% relative integration. (B) SRα RNA was translated in vitro and the membrane association of the resulting protein analysed as described in (A). (C) The membrane integration of Syb2 into various RM preparations was performed as described in (A), except that the RMs were reconstituted with in vitro-synthesised SRα in place of the recombinant protein used in panels A and B. SRP54 release indicates the percentage reduction in SRP54 adduct formation caused by the addition of the various RM preparations. In this case, the mean and standard error of two independent experiments are shown. (D) The membrane integration of Syb2G into various RM preparations was established as described in (C), except that the level of N-glycosylation was assayed in place of the amount of material resistant to sodium carbonate extraction. (E) The membrane integration of Sec61β was determined as described in (C). (F) The membrane integration of Cytb5C was determined as described in (C).

Figure 6

Membrane insertion of Syb2 is SRP dependent. (A) Syb2 or Syb2G was released from isolated RNCs by puromycin treatment in the presence or absence of GTP and SRP, and then incubated with SRP-depleted membranes for 10 min. With Ii123, the isolated RNCs were incubated with SRP-depleted membranes for 10 min before the puromycin treatment, and the incubation continued for a further 10 min after puromycin treatment in order to mimic cotranslational targeting. Membrane integration was assessed by extraction with sodium carbonate solution and quantification of Syb2 (upper panel), Syb2-1g product (middle panel) or Ii123-2g product (lower panel). Lane 1 shows the level of background signal obtained in the absence of any added membranes, and is set at 0% relative integration, as indicated in the legend to Figure 5A. (B) Sec61β was released from isolated RNCs by puromycin treatment in the presence or absence of GTP and SRP, and then incubated with SRP-depleted membranes for 10 min. Membrane integration was determined as described for Syb2 in (A). (C) Syb2 integration was assayed as for (A), except that the ‘after-release' sample was treated with puromycin before the addition of SRP and GTP. (D) Syb2 integration (upper panel) was assayed as for (A), except that ribosomes were removed by centrifugation prior to incubation with membranes. In one case (lane 4), an equivalent amount of purified, nonprogrammed, ribosomes was added back. To allow for potential losses of material during centrifugation, a fraction of the total products wase analysed and used to normalise the quantification of inserted Syb2 (loading control).

Figure 7

Unifying model for TA protein targeting to the ER membrane. Upon termination of translation, the insertion sequence of the TA protein emerges from the ribosomal exit tunnel, where an interaction with SRP is favoured by its proximity. If SRP remains bound, the TA protein can be targeted to SR at the ER membrane, allowing the possibility of its coordinated transfer to the Sec61 translocon for membrane insertion. Alternatively, where SRP fails to bind the TA protein, or binds and then becomes dissociated, a complementary ATP-dependent pathway can deliver the precursor to the ER membrane. The ATP pathway may deliver the TA protein to the Sec61 complex and/or novel component(s) for membrane insertion.