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Elongation arrest is a physiologically important function of signal recognition particle

2000; Springer Nature; Volume: 19; Issue: 15 Linguagem: Inglês

10.1093/emboj/19.15.4164

1460-2075

Nicola A. Mason, Leonora F. Ciufo, Jeremy D. Brown,

RNA Research and Splicing

Article1 August 2000free access Elongation arrest is a physiologically important function of signal recognition particle Nicola Mason Nicola Mason Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Swann Building, The King's Buildings, Mayfield Road, Edinburgh, EH9 3JR UK Search for more papers by this author Leonora F. Ciufo Leonora F. Ciufo Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Swann Building, The King's Buildings, Mayfield Road, Edinburgh, EH9 3JR UK Search for more papers by this author Jeremy D. Brown Corresponding Author Jeremy D. Brown Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Swann Building, The King's Buildings, Mayfield Road, Edinburgh, EH9 3JR UK Search for more papers by this author Nicola Mason Nicola Mason Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Swann Building, The King's Buildings, Mayfield Road, Edinburgh, EH9 3JR UK Search for more papers by this author Leonora F. Ciufo Leonora F. Ciufo Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Swann Building, The King's Buildings, Mayfield Road, Edinburgh, EH9 3JR UK Search for more papers by this author Jeremy D. Brown Corresponding Author Jeremy D. Brown Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Swann Building, The King's Buildings, Mayfield Road, Edinburgh, EH9 3JR UK Search for more papers by this author Author Information Nicola Mason1, Leonora F. Ciufo1 and Jeremy D. Brown 1 1Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Swann Building, The King's Buildings, Mayfield Road, Edinburgh, EH9 3JR UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:4164-4174https://doi.org/10.1093/emboj/19.15.4164 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Signal recognition particle (SRP) targets proteins for co-translational insertion through or into the endoplasmic reticulum membrane. Mammalian SRP slows nascent chain elongation by the ribosome during targeting in vitro. This ‘elongation arrest’ activity requires the SRP9/14 subunit of the particle and interactions of the C-terminus of SRP14. We have purified SRP from Saccharomyces cerevisiae and demonstrated that it too has elongation arrest activity. A yeast SRP containing Srp14p truncated at its C-terminus (ΔC29) did not maintain elongation arrest, was substantially deficient in promoting translocation and interfered with targeting by wild-type SRP. In vivo, this mutation conferred a constitutive defect in the coupling of protein translation and translocation and temperature-sensitive growth, but only a slight defect in protein translocation. In combination, these data indicate that the primary defect in SRP ΔC29 is in elongation arrest, and that this is a physiologically important and conserved function of eukaryotic SRP. Introduction Signal recognition particle (SRP) is the most widespread dedicated protein targeting factor, with homologues in all three kingdoms (Walter and Johnson, 1994; Lütcke, 1995; Bui and Strüb, 1999). It plays a crucial role in ensuring targeting of presecretory and membrane proteins to the eukaryotic endoplasmic reticulum (ER), bacterial cytoplasmic and chloroplast thylakoid membranes. Canine SRP, the first identified, was purified on the basis of its translocation-promoting activity (Walter and Blobel, 1980). It binds tightly to ribosome–nascent chain complexes in which signal sequences of ER-targeted proteins are just exposed (Siegel and Walter, 1988a). This binding results in a slowing of translation, a phenomenon termed ‘elongation arrest’ (Walter and Blobel, 1981). This is proposed to increase the window of opportunity for the SRP–ribosome–nascent chain complex to interact with the SRP receptor in the ER membrane, the next step towards docking the ribosome on to the translocation apparatus and establishment of co-translational translocation. SRP is a ribonucleoprotein consisting, in higher eukaryotes, of the 7SL RNA and six proteins. All subunits are required for full activity of SRP, but particular activities have been ascribed to individual components (Siegel and Walter, 1985, 1988b; Krieg et al., 1986; Kurzchalia et al., 1986). Elongation arrest requires the SRP9 and SRP14 proteins that bind as a heterodimer to the 5′ and 3′ ends of the 7SL RNA in the ‘Alu-domain’ of the particle (Strüb et al., 1991). Recent work (Birse et al., 1997) has elaborated the three-dimensional structure of SRP9/14; the complex is symmetrical, the two proteins adopting the same fold despite dissimilar primary sequences. SRP lacking the Alu-domain or just SRP9/14 can promote translocation but lacks elongation arrest activity (Siegel and Walter, 1985, 1986). These particles also have reduced affinity for ribosomes lacking signal sequences (Hauser et al., 1995; Powers and Walter, 1996). In contrast, SRP reconstituted with an SRP14 lacking 20 C-terminal amino acids (ΔC20) lacks elongation arrest activity but retains its affinity for ribosomes (Thomas et al., 1997). This mutant thus defines an elongation arrest-specific SRP–ribosome interaction. Elongation arrest was first observed as a halt in translation of signal sequence-containing proteins by wheat germ ribosomes mediated by canine SRP (Walter and Blobel, 1981). In a mammalian translation system, however, canine SRP only delays production of full-length translation products (Wolin and Walter, 1989). All elongation arrest-deficient SRP derivatives have decreased translocation-promoting activity (Siegel and Walter, 1985, 1986; Hauser et al., 1995; Thomas et al., 1997), suggesting that elongation arrest is important for full SRP activity. However, mammalian SRP is the only eukaryotic SRP studied and it is therefore unknown whether elongation arrest is peculiar to it, or is a conserved function. As all studies have been undertaken in vitro, the physiological importance of elongation arrest has not been addressed. Here we examine functions and interactions of SRP in the yeast Saccharomyces cerevisiae, an organism amenable to biochemical and genetic analysis. SRP is not essential for growth of yeast cells, although it is required for efficient ER targeting of proteins that contain strongly hydrophobic signal sequences (Ng et al., 1996), and in its absence yeast cells grow poorly (Hann and Walter, 1991). Yeast SRP contains at least six proteins (Srp72p, Srp68p, Srp54p, Sec65p, Srp21p and Srp14p), and its RNA is scR1 (Hann and Walter, 1991; Hann et al., 1992; Stirling and Hewitt, 1992; Brown et al., 1994). The proteins are homologous to those of mammalian SRP, with one exception, Srp21p. No Srp9p has been identified in yeast SRP, although a 7 kDa protein was found in fractions containing purified yeast SRP proteins (Brown et al., 1994). Recently, recombinant yeast Srp14p was shown to homodimerize and bind to RNAs containing the 5′ portion of scR1 (Strüb et al., 1999), suggesting a novel Alu-domain structure. Here we present purification of active yeast SRP, the first characterization of a eukaryotic SRP other than the canine particle. It contains only the six proteins previously determined to be in it and no SRP9 homologue. However, it has two Srp14p subunits. Yeast SRP has elongation arrest activity, and interactions of the C-terminus of Srp14p, crucial for elongation arrest in mammalian SRP, are vital for this activity in this particle both in vitro and in vivo. Elongation arrest is therefore a conserved, functionally important feature of eukaryotic SRP. Results Purification of yeast SRP We constructed a yeast strain expressing Srp72p extended at its C-terminus by a cleavage site for the tobacco etch virus (TEV) protease and two copies of the protein A immunoglobulin (Ig) binding domain (see Materials and methods). The modified protein fully complemented a lack of endogenous Srp72p and we isolated SRP from a post-ribosomal extract of this strain on IgG–Sepharose, eluting it with TEV protease. One further purification step yielded fractions (Figure 1A, lanes 8 and 9) that contained almost exclusively proteins of the sizes of known yeast SRP components, and immunoblotting confirmed that they were Srp72p, Srp68p, Srp54p, Sec65p, Srp21p and Srp14p (data not shown). Purified SRP did not contain a 7 kDa band as found previously in pools of SRP proteins (Brown et al., 1994). This, and the lack of a yeast open reading frame encoding an SRP9 homologous protein, indicates that yeast SRP does not contain SRP9. We also purified SRP containing Srp14p lacking the last 29 amino acids (ΔC29; Figure 1B). This truncation mimics the ΔC20 mutation, which dissociates ribosome binding and elongation arrest activities of mammalian SRP14 (Thomas et al., 1997), and we hoped that it would provide insight into these functions of the yeast particle. Figure 1.Purification of yeast SRP. (A) SRP was purified as described in Materials and methods, fractions resolved on a 10% Proseive (FMC Bioproducts) gel and stained with Coomassie Blue. Lanes 1 and 2: post-ribosomal extract and IgG–Sepharose flowthrough (1/16 000); lanes 3–5: TEV eluate, ω-aminobutyl agarose flowthrough and wash (1/80); lanes 6–12: elutions (1/20). Marker and SRP proteins are indicated. (B) Purification of SRP ΔC29 as in (A). ΔC29 contains an N-terminal triple-HA tag and thus runs above Srp21p. (C) RNA analysis. RNA isolated from purified SRP was run on a 6% acrylamide–50% urea gel and stained with ethidium bromide. Lane 1: pBR322 MspI digest markers; lanes 2 and 3: RNA from fractions shown in lanes 8 of (A) and (B), respectively (1/20). Download figure Download PowerPoint Analysis of purified SRP for RNA revealed a prominent species (Figure 1C, lanes 2 and 3) of a size consistent with the 519 nucleotide length of scR1 (Felici et al., 1989; Hann and Walter, 1991). Northern blotting confirmed that it was scR1 (data not shown). Purified yeast SRP is active We examined the ability of purified SRP to promote translocation of SRP-dependent substrates into ER-derived microsomes. The synthetic mRNA species used to programme these translations encoded the SRP- dependent chimeric protein DHCαF (Ng et al., 1996) and prepro-α-factor (ppαF), which is targeted to the yeast ER independently of SRP. We also used preprolactin (ppL), used extensively in characterization of canine SRP. The ppL used contains a single amino acid substitution, glycine–alanine, at amino acid 10. This increases the efficiency of secretion of a protein containing the ppL signal sequence from yeast (Ngsee and Smith, 1990) and the translocation of ppL across the yeast ER (J.D.Brown, unpublished data). The identity of translocated species produced from these substrates was verified by virtue of their being protected from digestion by protease (Figure 2A). Figure 2.Activity of purified yeast SRP. (A) Confirmation of translocated species by protease protection. Translation reactions were carried out with or without addition of microsomal membranes (yRM) and subsequently incubated with 0.5 mg/ml proteinase K where indicated. (B and C) Translation reactions were carried out using extract containing (+) or immunodepleted of (−) SRP. yRM and purified SRP were added as indicated (concentrations of SRP in nanomoles). SRP was wild type except in (B) lane 13 and (C) lane 9, where SRP ΔC29 was used. (D) Translation reactions contained non-depleted extract supplemented with yRM and SRP (100 nM) as indicated. Reactions were run on 15% SDS–polyacrylamide gels and visualized by autoradiography. Untranslocated (ppL, ppαF and DHCαF) and processed, translocated (pL, gpαF and gDHCαF) species are indicated. Graphs were plotted using averaged data from three independent experiments. The translocation with non-depleted extract was set as 100%, bars, 1 SD. Download figure Download PowerPoint SRP-depleted cytosol (Ng et al., 1996) was substantially defective in promoting translocation of DHCαF and ppL (Figure 2B, lane 7 and C, lane 3, respectively) but translocation of ppαF was unaffected (Figure 2B, lane 3). Addition of purified SRP restored translocation of DHCαF and ppL in a dose-dependent manner (Figure 2B, lanes 8–12 and C, lanes 4–8, and graphs) and it was thus active. Immunoblotting and comparison with known amounts of purified SRP allowed us to estimate the concentration of SRP in translation reactions containing non-depleted cytosol to be 10 nM (data not shown). Addition of 10 nM SRP to depleted cytosol restored translocation to ∼75% of that seen with non-depleted extract (Figure 2B and C graphs), and purification had therefore not substantially impaired the activity of SRP. SRP containing ΔC29 had less translocation-promoting activity than the wild-type particle. Addition of 100 nM mutant particle to SRP-depleted cytosol resulted in translocation of DHCαF or ppL similar to that achieved with 2–5 nM of the wild-type particle (Figure 2B and C, lanes 11–13 and 7–9, respectively). One interpretation of this result is that the mutant particle was largely defective in binding ribosome–nascent chain complexes. We therefore performed experiments (Figure 2D) in which we added either wild-type or ΔC29 SRP to reactions that contained non-depleted cytosol. Wild-type SRP increased translocation of DHCαF and ppL (Figure 2D, lanes 4 and 8 and graph). However, the mutant SRP decreased translocation of these proteins (lanes 3 and 7) but not ppαF (data not shown). The simplest explanation of this result is that the mutant SRP competed with the endogenous SRP for binding available ribosome–nascent chain complexes, but in most cases failed to target them efficiently to the membrane. Thus, the SRP ΔC29 is not grossly deficient in ribosome–nascent chain binding. Yeast SRP has elongation arrest activity An explanation for the reduced translocation-promoting activity of the ΔC29 mutant is that it lacks elongation arrest activity and therefore cannot retain nascent chains in a short translocation competent state. We tested yeast SRP for elongation arrest activity in time-course experiments comparing the kinetics of production of full-length translation product in the presence or absence of added SRP. We used DHCαF as an SRP-dependent substrate and ppαF as a negative control. The comparison between these two substrates was particularly useful as they are of similar length and composition, differing chiefly in the signal sequence (Ng et al., 1996). Wild-type SRP reproducibly delayed production of full-length DHCαF by 1–2 min; it was just detectable at 4 min without addition of SRP, and at 6 min when SRP was added (Figure 3A, compare lanes 4, 5 and 6). In contrast, full-length ppαF was produced with similar kinetics whether SRP was added or not, appearing after 4 min (Figure 3A, lane 12). In equivalent experiments with SRP containing ΔC29 (Figure 3B), we detected no delay in the production of either DHCαF or ppαF, suggesting that this mutant was indeed defective in elongation arrest. Quantification and averaging of data from three experiments (Figure 3C and D) confirmed that addition of wild-type SRP, but not SRP containing ΔC29, delayed the initial appearance of full-length DHCαF, but not its rate of accumulation. Figure 3.Yeast SRP mediates elongation arrest. (A and B) In vitro translation reactions were carried out as in Figure 2 using mRNAs encoding DHCαF or ppαF (left and right panels, respectively) with or without 100 nM added SRP. SRP was wild type (A) or ΔC29 (B). Samples were stopped at the times indicated. All panels are from the same experiment, and are equivalent exposures processed identically. (C and D) Quantified data from three experiments performed with DHCαF were averaged and plotted. SRP was wild type (C) or ΔC29 (D). Squares, plus SRP; diamonds, without added SRP. Download figure Download PowerPoint To demonstrate further the significance of the effect of SRP on translation, we carried out 40 translation reactions programmed with DHCαF mRNA, of which half contained additional SRP. For each condition 10 reactions were stopped at 7 min, when the reaction is in its linear phase of product accumulation and 10 were stopped at 30 min when the maximum amount of product has been produced (data not shown). Quantification of reaction products and analysis by t-test confirmed that at 7 min there was a significant (98% confidence level) difference in the levels of product between reactions, which were lower with added SRP than without it (see Materials and methods). At 30 min there was no significant difference, and SRP had no effect on the total product made. Analogous experiments using mRNA encoding ppαF revealed no statistically significant difference in the amount of translation product in the presence of additional SRP. Thus, yeast SRP does indeed promote elongation arrest during translation of DHCαF mRNA. In equivalent experiments using SRP containing ΔC29 we could, as in the single time-course experiment, detect no difference in the translation of either DHCαF or ppαF. Yeast SRP contains two Srp14ps Conservation of elongation arrest but lack of SRP9 in yeast SRP prompted us to investigate its composition. In particular we analysed the copy number of Srp14p and other proteins in the particle, using strains carrying genes encoding two different copies of the protein of interest. For Srp14p we used a strain containing a fully functional (see Figures 5,6,7) triple haemagglutinin (HA) epitope-tagged Srp14p and the untagged protein. Immunoprecipitation of the two Srp14p variants with antibodies against Sec65p from an extract of this strain indicated that they were both efficiently assembled into SRP (Figure 4A, lanes 4 and 5). Immunoprecipitation with anti-HA antibodies, under conditions where almost all the tagged protein was bound by antibody, resulted in co-isolation of approximately half the wild-type protein (Figure 4A, lanes 2 and 3). This is consistent with the particle containing two Srp14p proteins, the two variants associating randomly, resulting in a 1:2:1 ratio of tagged only:tagged plus untagged: untagged only. SRP was not immunoprecipitated with anti-HA antibodies when there was no tagged Srp14p in the extract (data not shown). Similar experiments with extracts from strains carrying two Srp72p or Srp21p variants did not result in co-isolation of both copies of either protein with reagents that bound only one (Figure 4B and C, lane 2). Thus, the particle is monomeric in cell extracts and contains one copy of each of these proteins. Taken together with the studies of Strüb et al. (1999), which demonstrated in vitro binding of a dimer of recombinant Srp14p to scR1 RNA, the conclusion is that the protein composition of the Alu-domain of yeast SRP differs from the equivalent domain of mammalian SRP, most likely containing a homodimer of Srp14p. Figure 4.Yeast SRP contains two Srp14ps. (A) Extracts of strains expressing either wild-type and HA-Srp14p (lanes 1–5) or just HA-Srp14p (lanes 6–10) were immunoprecipitated with anti-HA (lanes 2 and 7) or anti-Sec65p antibodies (lanes 4 and 9). Material corresponding to 50% used in each immunoprecipitation (lanes 1 and 6) and of each supernate (lanes 3, 5, 8 and 10) and all immunoprecipitated material were analysed. (B and C) As (A) except that strains used expressed protein A-tagged Srp72p and wild-type Srp72p (B) or Myc-Srp21p and HA-Srp21p (C). Proteins were isolated using IgG–Sepharose or the antibody indicated. *, Ig chains recognized by secondary antibodies. Download figure Download PowerPoint Figure 5.Truncation of Srp14p results in delayed onset temperature sensitivity. (A and B) Strains expressing HA-Srp14p (open squares), DC11 (diamonds), ΔC29 (triangles) or lacking Srp14p (squares with crosses) were grown in liquid culture at either 23 (A) or 36°C (B). (C) sec65-1 cells were grown at 23°C and half transferred to 36°C (arrow). Download figure Download PowerPoint Figure 6.SRP containing ΔC29 is stable at 36°C. (A and B) Protein or RNA samples were prepared from cells expressing Srp14p variants grown at 23°C or following transfer to 36°C for the time indicated. (A) Equal amounts of protein samples (assessed by Bradford assay) were analysed by imunoblotting. (B) Equal amounts of RNA (assessed by measuring OD260) were denatured, electrophoresed through a 6% acrylamide–50% urea gel, blotted on to a nylon membrane and hybridized sequentially with 32P-labelled DNA fragments of SCR1 and SNR19. 36°C samples were taken after 16 h growth at this temperature. (C) Whole-cell extracts from strains grown at the temperatures indicated for 16 h were prepared and fractionated on 10–30% w/v sucrose gradients. One quarter of each gradient fraction and 1/10th load were analysed for SRP proteins. In this experiment, SRP in srp14-ΔC29 strain extracts sedimented at slightly different rates. This was not seen in other experiments. Download figure Download PowerPoint Figure 7.Cells expressing ΔC29 have slight translocation defects for Pho8p, but not DPAPB or CPY, at 36°C. Immunoprecipitations were carried out with antibodies against the indicated proteins from extracts of cells pulse-labelled for 6 min (see Materials and methods). Immunoprecipitates were electrophoresed through 10% Proseive gels and visualized by autoradiography. Labelling was at 23°C (srp14-ΔC29, srp14), 30°C (sec63-201) or following transfer from 23 to 36°C for 30 min (sec65-1) or 16 h (srp14-ΔC29, HA-SRP14). Dipeptidyl aminopeptidase B: DPAPB, glycosylated luminal; pDPAPB, cytoplasmic. Alkaline phosphatase: Pho8p, glycosylated luminal; pPho8p, cytoplasmic. Carboxypeptidase Y: gpCPY, glycosylated lumenal; ppCPY, cytoplasmic. In this experiment a band appeared in the sec63-201 sample at the position of pDPAPB. This was not seen in other experiments. Download figure Download PowerPoint Cells expressing Srp14p-ΔC29 are temperature sensitive Our in vitro data are consistent with the primary defect of the ΔC29 mutant being in elongation arrest. The competition experiments (Figure 2D) indicated that it bound ribosome–nascent chain complexes, but we could not exclude the possibility that it was partially defective in this activity. However, since strains expressing the mutant grew well in culture at 30°C (data not shown), readily allowing us to produce sufficient cells for purification of the particle, we considered that the severe targeting defects of the purified ΔC29 particle in vitro may be an exaggeration of the in vivo phenotype. We therefore took advantage of the yeast system and studied the mutant in vivo to see if we could obtain a more accurate measure of its defects. Srp14-ΔC29 cells grew poorly on plates at 36°C (data not shown) and we tested their growth in liquid culture. At 23°C, srp14-ΔC29 cells grew at the same rate as wild type and those expressing full-length HA-tagged Srp14p, but slowed and stopped growing after ∼16 h at 36°C (Figure 5A and B and data not shown). This contrasted dramatically with the almost immediate growth defect of the only previously published thermo-sensitive SRP mutant, sec65-1 (Figure 5C). However, a yeast strain carrying an srp14 deletion showed a similar growth profile to srp14-ΔC29 cells (Figure 5B). This conditional growth defect of yeast lacking an SRP component had not been noted previously, and to ensure that this was not specific to srp14 mutants we examined growth of strains lacking other SRP subunits. These strains were similarly temperature sensitive, ceasing growth at 36°C after 16–20 h (data not shown). ΔC29 is stable and SRP associated Next we studied the amount and integrity of SRP in ΔC29 cells. This would determine whether the slowing of growth of srp14-ΔC29 cells at 36°C was due to newly synthesized SRP or ΔC29 protein being unstable, resulting in decreased SRP activity over time. We compared the level of Srp14p-ΔC29 with full-length protein and that of a truncation, ΔC11, which lacks a yeast-specific extension to Srp14p but does not affect growth (Figure 5A and B). The amounts of the various Srp14ps did not change dramatically after transfer to 36°C, even beyond times at which growth of srp14-ΔC29 cells was seriously affected (Figure 6A, lanes 1–12). Srp14p-ΔC29 was therefore stable. Next, we examined scR1 since lack of Srp14p results in a dramatic decrease (10- to 20-fold) in its steady-state level (Brown et al., 1994). The amount of scR1 RNA remained relatively constant at 36°C (Figure 6B, lanes 2–5), suggesting that ΔC29 was not only present but also associated with SRP at 36°C, and we tested this directly by performing sucrose density gradient sedimentation on cell extracts. In extracts of srp14-ΔC29 cells grown at 23 or 36°C for 16 h (when growth begins to slow), virtually all the Srp14p co-sedimented with another SRP protein, Srp54p (Figure 6C). Thus, the growth defect of srp14-ΔC29 cells cannot be accounted for by loss of SRP, but is rather a consequence of a functional defect in the mutant particle. Translocation defects associated with ΔC29 We examined srp14-ΔC29 cells for defects in targeting to the ER membrane. If the decreased translocation in in vitro assays containing the ΔC29 particle were due to overall decreased activity then we expected this to be revealed in vivo through the persistence of cytosolic forms of SRP-targeted proteins lacking ER-specific signal sequence cleavage and/or glycosylation. We pulse-labelled srp14-ΔC29 cells with [35S]methionine/cysteine following growth at either 23 or 36°C for 16 h, and immunoprecipitated proteins from cell lysates (see Materials and methods). srp14-ΔC29 cells had no defects for any of the substrates tested at 23°C (Figure 7, lane 1), only ER-modified forms being visible. At 36°C a slight defect was revealed in the translocation of the SRP-dependent substrate Pho8p compared with cells expressing full-length HA-tagged Srp14p, a small amount of non-glycosylated precursor being visible (Figure 7, compare lanes 2 and 3). We did not, however, see a defect for DPAPB, typically severely affected in cells compromised for SRP activity. Control strains gave the expected results. Thus, the sec65-1 mutant revealed a virtually complete failure to translocate both Pho8p and DPAPB (Figure 7, lane 6), and an srp14-null strain revealed partial translocation defects for these proteins (Figure 7, lane 4). Such attenuated defects are typical of cells permanently lacking SRP function, and are the result of adaptive changes that occur in such strains (Ogg et al., 1992). A strain compromised for the SRP-independent pathway (sec63-201; Ng et al., 1996) had the expected severe translocation defect for CPY (Figure 7, lane 5). The results obtained with the srp14-ΔC29 mutant were striking. It does not have severe defects in SRP-dependent targeting and we concluded that SRP containing Srp14p-ΔC29 must bind ribosome–nascent chain complexes efficiently and target them to the ER membrane in vivo. The limiting factor in co-translational targeting is translation beyond the point at which SRP can no longer remain ribosome bound (Siegel and Walter, 1988a). Therefore, if the primary defect in ΔC29 is in elongation arrest rather than signal sequence recognition, and for the substrates tested (Pho8p and DPAPB) this ‘point of no return’ was passed occasionally, then slight translocation defects, such as those seen, would result. We therefore employed an alternative measure of targeting by SRP in vivo, in particular one that could probe elongation arrest activity. This was the ubiquitin-assisted translocation assay (UTA) (Johnsson and Varshavsky, 1994), specifically designed to measure the coupling of translation and translocation. UTA reporters (Figure 8A) encode a signal sequence followed by a variable length of ‘spacer’, then ubiquitin and finally a marker, the cytosolic enzyme Ura3p in the reporters used here. Close coupling of translation and translocation results in the whole protein being translocated into the ER, and the cells remain ura– in an otherwise ura3 genetic background (Ura3p is required in the cytosol to function). If targeting is not efficient or closely coupled to translocation, the ubiquitin moiety is exposed in the cytosol, folds rapidly, and ubiquitin-dependent proteases cleave C-terminally to it leaving Ura3p in the cytosol—cells are then ura+. Figure 8.Translocation defects of srp14-ΔC29 cells in the UTA assay. (A) Organization of UTA constructs. The protein is depicted N- to C-terminus; signal sequence (SS), spacer (SPACER), ubiquitin (UBI) and Ura3p. The cleavage site for cytosolic ubiquitin-dependent proteases is indicated (arrow). (B and C) srp14-ΔC29 cells streaked on to a −trp (B; selecting for the plasmids) and a −ura (C; selecting for cytosolic Ura3p) plate were incubated for 2 days at 30°C. They contained empty vector (top left), Suc2277 (top right), Dap2300 (bottom left) or the control substrate Suc223, which has a 23 amino acid spacer (bottom right). (D and E) As for (B) and (C) but with wild-type cells. As seen previously (Johnsson and Varshavsky, 1994), Suc223 gave a Ura+ phenotype in wild-type cells. (F) Anti-HA immunoprecipitations from extracts of pulse-labelled cells expressing Suc2518UbDHFRHA (lanes 1–3) or Suc223UbDHFRHA control, which only yields the DHFRHA fragment (lane 4) (Johnsson and Va