ARTICLES A protein complex required for signal-sequence-specific sorting and translocation Brigitte Wiedmann’, Hideaki Sakai, Terri A. Davis & Martin Wiedmann 1, Now York, New York 10024, USA Cellular Blocnemistry and Biophysics Program, Memoral Sloan-Kettering Cancer Cer We have purified a nascent-polypeptide-associated complex (NAC) which prevents short ribosome-associated nascent polypeptides from inappropriate interactions with proteins in the cytosol. NAC binds nascent-polypeptide domains emerging from ribosomes unless a signal peptide is fully exposed. Depletion of cytosolic proteins (including NAC) from ribosomes carrying nascent polypeptides allows the signal recognition particle (SRP) to crosslink to polypeptides irrespective of whether or not they contain signal peptides. In the absence of cytosol, proteins lacking signal peptides can be mistranslocated into the endoplasmic reticulum in vitro, albeit with low efficiency. Readdition of NAC restores the specificity of SRP and fidelity of translocation. SEVERAL proivins are known to interact with ribosome-associ ated nascent chains (hereafter referred to simply as nascent chains), Proteins such as the chaperones or modifying enzymes (for example, signal peptidase, oligosaccharyl transferase or pro- {ein disulphide isomerase) can interact with both nascent chains sand polypeptides that have been released froma the ribosome! Here we describe the identification and purification of a nascent polypeptide-associated complex (NAC). a heterodimeric protein ‘which binds to short nascent chains but not to released chains, with the exception of those with fully exposed signal peptides Using in vitro assay systems lacking most cytosolic factors (including NAC) that bind nascent chains in a sal-sensitive manner, we show that signal recognition particle (SRP) can interact with proteins lacking signal peptides. SRP. which acts as.a shuttle between the cytosol and the endoplasmic reticulum (ER) membrane, is the only other known “non-ribosomal’ factor edical 10028, USA, Deparinent of Call ‘which interacts exclusively with nascent, as opposed to released, chains. When bound to signal peptides, SRP, by interacting with ity receptor at the ER membrane, mediates the targeting and transport of nascent chains to and across the ER membrane” ‘We now show that SRP can mediate the targeting to and trans focation across the ER membrane of proteins lacking signal peptides, Readdition of purified NAC to eytosollepleted ribosomes both restores the specificity of SRP binding and prevents mis largeting and therefore inappropriate translocation. We hypothesize that, as NAC is likely 10 be one of the first non Hibosomal proteins with which a nascent chain makes contact, it functions in part to shield non-signal peptide regions of nascent chains from potentially promiscuous interactions with SRP. Identification of NAC To identify proteins interacting with nascent chains, we trans: lated truncated messenger RNAs encoding different proteins and lacking stop codons im vitro to produce stable ribosome. associated nascent-chain intermediates of delined length(s)" NATURE VoL 370 + 11 AUGUST 1994Additionally, instead of lysine, Iysyl residues derivatized with the photoactivatable crosslinker TDBA. (4-(3-trifluoromethyl- diazirino)benzoic acid) were incorporated at kaown positions, These nascent chains bearing photoactivatable probes are sub- strates for naseent-chain-binding proteins, and photoactivation causes covalent attachment of interacting partners, The highly reactive carbenes produced on photoactivation ofthis very short probe have a halflife of about Ins and can react with any chemical bond’, thus allowing control of spatial and temporal conditions of crosslinking. Following sodium dodecy! sulphate~ polyacrylamide gel electrophoresis (SDS-PAGE) and Muoro- ‘eniphy, the size of the crosslinked protein is estimated by sub- Lracting the relative molecular mass (M,) of the naseent chain from the sizeof the photoadluet’. The nascent chains used here are: peroxisomal firefly luciferase (Mu), mitochondrial pre-F! ATPase f-subunit (pF'B), chloramphenicol accty! transferase (CAT), ‘mitochondrial pre-cytochrome oxidase IV subunit (PCOXIV), bovine preprolactin (pPL) and a deseribed signal Peptide mutant of pPL (pPL-M) that is not translocated*. The munber of translated amino acids (aa) precedes the name of each nascent chain, Figure la shows that nascent chains of S2aaffLuc, saffLuc, SSaapPL-M and SlaapF'f can be crosslinked to sev- 1 proteins present in the wheat-germ eytosol, with a crosslink 10 4 protein of :M, ~33,000 (33K) being predominant (see Fig, la legend for positions of lysines and methionines). To determine whether or not the crosslinked protein is a core ribosomal protein, we treated the nascent chains with inereasing salt concentrations before sedimentation through salt/sucrose cushions and subsequent irradiation. As shown in Fig, Id (anes 1-5), the 33K protein is bound to ribosome-associated nascent chains in a salt-sensitive manner. Puromycin release of the nascent chain before irradiation abolished the crosslink, stg gesting that the 33K protein only binds polypeptides in the eon- text of the ribosome. Puromycin treatment after irradiation released the crosslinked product from the ribosome (not shown, but see Fig, 26), supporting the view that the 33K protein is not core ribosomal protein, To investigate whether this protsin binds reversibly, nascent chains were extracted with high salt and isolated by sedimenta- tion through a salt/suerose cushion, The 33K. protein was crosslinked when eytosol from wheat germ (Fig. 1A, lanes 6, 7), bovine brain (lanes 8, 9) and dog pancreas (lanes 10, 11) was added to the bare nascent chains before irradiation. Therefore, the 33K protein is salt-extractable, ean rebind after extraction and is present in a variety of species and tissues, Purification of NAC We used salt-stripped S2aaffLue nascent chains as the substrate for purifying the 33K protein from bovine brain cytosol (Fig 2a). Throughout our purification, a 33K protein eo-purified with a 21K protcio which is also evident but net dominant in crude cytosol (see Fig. 1a), These proteins, named aNAC and BNA( form a heterodimeric complex (H.S. et a., manuscript in prepa’ ation), Both subunits could be crosslinked to the nascent chain (Fig. 25 and ¢}. Puromycin addition before crosslinking pre- vented purified NAC from binding to nascent chains (Fig. 24) and its addition after crosslinking released crosslinked NAC along with the nascent chain (data not shown), Peptide sequence data from purified a-and {NAC and subse quent complementary DNA cloning of the human genes indi- ‘ated the following homologies: @NAC is homologous to three overlapping DNA fragments of unknown function whieh encode all but the amino-terminal 23 amino acids of NAC (GenBank accession numbers D12194, Z28479 and 225168)". BNAC is identical to BTF3b, a protein of unknown function’! ‘To prove that the proteins we purified are indeed the ones that give tise to the crosslinks, peptide-specific antibodies to the deduced carboxy-terminal amino-acid sequences of a= und ANAC were generated and used to immunoprecipitate the ‘erosslinked nascent chains (Fig, 2c) 41 AUGUST 1994 NATURE - VOL 370 a oF oe ae ‘roa * * a FIG. A33K protein present in the cytosol of venous tissues can bind to short nascent chains in a salt-sensiive manner. 2, Crossinking In ssheat germ Iysate. xv" indicates irradiation and the asterisk marks the crosslinking product between the 33K protein and the nascent ‘chains. &, The 33K protein binds in a saltserskive manner and is resent in various cel types. The crosslink between 52aaffLue nascent ‘chains and the 33K protein lane 2) s abolished when nascert chains were adjusted 19 500 mM potassium acatate (H) and sedimented trough sucrose cushions, Retuen of cytosol from wheat getm (lanes 6, 7), bovine brain (lanes 8, 9) or dog pancreas (lanes 10, 14) leads (9 the reappearance of the erassink. METHODS. a, In vitro translation of truncated mRNAS and photo: crosslinking are as described elsewhere™™. Samples were treated wth RNase A, preciptated with TCA and visualized by fluorography after ‘SDS-PAGE. The postions of lysines and methionines in the proteins are given: ysines Methionines Ta flue 6,8,9,28, 31,67 Gap, 6aanPLM 4,9, 72,78 ‘962ap00X 12, 28, 32,52,78, 85,92 Stop! 16, 19, 30, 45 Teaacat 3,4,19,46,49,50,54 1, 66, 75 », After transition, aliquots wore adjusted to 150 mM (L), 250 mM (i ‘oF 500 mM ¢H) potassium acetate and spun through U.5 M suerase Cushions in TSB translation mixture lacking lysate, radiolabel ‘and ‘nRNA| ofthe same potsssium acetate concentration (20 min, TL120.1 fotos, Beckman; 120,000 r.p.m. 4 vol. sample over 3 vols cushion) Pel: lets were resuspended in a haltvolume of TBB, Aliquots (7 il} were either iradiated immediately and processed as desorined above lanes 3-5)0r processed after Incubation for 5 min on ke with 1 or yl cytosol (WG: ge fitrated, 20,000g supernatant, 30 mgrn\ "; BBC: gl rated, 100,000g, supematant, 25mg mi; UPC: 1000008 supematant 15 mgm *) 438,ARTICLES b © S2aa fue strona anirmiue |= + = Hecoste proimmuno | => 42 22> 70 anteonac [>> 74 42> i antepwac, |= 222 fae 030 eSmprepide| > 23 tt 460-4 3004 143 : 129456 Mik) Je FIG. 2 Purification and patial characterization of NAC. a, Protein pat ‘orn at diferent stages of purcation after SDS-PAGE and Coomassio hie staining. Lane 4, bovine brain cytosol, dialed with 30-60% ‘ammanium sulphate fraction, 16 lane 2, supernatant after heating, Bye: lane 3, QSepharase FF active faction, 15 ye: lane 4, RedA Matrex active traction, 12 yg: lane 5, Heparin-Hitrap active fraction, 4g lane 6, Mono 0 active fraction, 2 ys Two proteins, NAC and [BNAG, ware Copurifed by this procedure. Crosslinks to NAG are indica fed in al figures by one (aNAG) or two (PNAC) asterisks. 0, Puriled NAC binds only to naseent-chain nbosome complexes. When nascent chains were released from the lbosomes, no crosslink to NAC is observed (lanes 2 and 4), c, The puriiod «NAC and fINAC proteins are identical ta the crosslinked proteins. S2aafLuc nascent chains were crosslinked te purified NAC and then eithar subjected to SOS-PAGE (lane 1) or Immunopreciptated wit fet luciferase (ane 2), preimmune (lane 3) ‘aNiAC anes 4 and 5) of NAC lanes 6 and 7) sora, The spoctcty of tech antibody was veried by competition with antigenic peptide (anes 5 and 7} METHODS, 2, Bovine brain was homogenized in 2 vols 250 mM sucrose in bulfer A (25 mM Tris-HCl pH8.0, 50.mM KCl, 1 mNt cithiotheltal (TT), mM EGTA, 5 mM MgCl, 1 mM phanyimethylsulphony| fluoride [PMSF Aer centrifugation (745, Beckman; 40.000 rpm. 1.51) a Giajzed 30-60% ammonium sulphate fraction of the supernetent wes heated for S min at 70°C and then spun as before, but for 2h. The Cleared supernatant was appliaé to a Q-Sepharose FF column (Phar ‘macia). The actty in each fraction was measured by crosslinking 10 NAC prevents inappropriate SRP crosslinking Because the ist lysine inthe SlaapF”B nascent chain is 16 amino acids from the N-terminus and this lysine crosslinks to the 3K protein (Fig, La) and to purified NAC (not shown), we conclude that NAC can bind regions of nascent chains as near as 35 amino acids from the peptidyl transferase. Thus, NAC is very near to ‘where the nascent chain leaves the ribosome (exit site). The SRP SAK protein has previously been shown to bind to the signal sequence when near the exit site!” ‘We have shown that in unfractionated eytosol, nascent chains lacking signal peptides primarily crosslink to NAC. whercas proteins with signal peptides predominantly crosslink to SRP (see Figs la and 30), The identity of the crosslink to SRP S3K ‘vas established. by immunopreeipitation (not shown). In the case of a very short preprolactin nascent chain SSaapPL), we served crosslinks to both SRP and NAC (Fig. 34, lane 13). Tt has been shown" that SRP-arrested preprolactin nascent, chains of 70 amino acids crosslink solely to SRP S4K. To deter mine directly whether NAC and SRP have different specificities, we prepared cytosobdepleted nascent chains of S6aapPL, TraalfLuc or SSaapPL and added SRP to each. Surprisingly, SRP crosslinked to all nascent chains regardless of whether oF not they harboured a functional signal peptide (Fig. 3a, lanes 4 436 149] ie wT 2s eer set stroped $2eamT.ue nascent chains, which gave major photoadducts| {of 42K and 27K. Fractions over 280-430 mM KC! were combined and then applies to a Red-A Matrex column (Amicon), After washing with ‘300 ent KCI n urler B(20 mM HEPES pH 7-5, 1. mA DTT. 1 mM PMSF, 25% glycera), the actvty was eluted with 1M KCI in buffer B. The fluent was diutee 10-fold with buflar 8 and applied to @ prapacked Henatin-HiTrap column (Pharmacia). The eluent at 400 mM KCI was. diluted 5-fold with buffer, applied to a Mone-0 column (Pharmacia) and eluted with a linear gradient of 100-800 mM KCI in buffer 8. & Single protein peak as ebtained at 320 mM KCI concentration which shows a single protein band in naive PAGE (not shown) and two Berd, of 33K and 24K Jn S0S-PAGE, a6 shown in lane 6. From 800g of bovine brain, 750 ue of purified NAC was obtained. b, Saltstiipped ascent chains of S2aatfluc ar SSaapPL were treated for 10 min at 30. Cwith mM puromycin and 100 yg mi” RNase A (lanes 2 and 4) OF mockttested (lanes 1 and 3). Purlfed NAC (200 nM) was allowed to bind for 5 min at 26 before iradiation.c, Immunopreciptation. SaatfLuc-NAC crosslink adducts wore teated with RNase A (400 sgt *) for 10 min at 30 "C10 eigest pepticyltransfer RNAS. The Samples were adjusted to 1° SDS and immunoprecipitated by a sta dard procedure”. NAC and NAG antibodies were raised in rabbits against synthetic peptides corresponding to the C-terminal sequence fa human expressed saquence tag HEST: GenBank accession number 12194), RALKNNSNDIVNAIMELTM-COOH, and of BTF3n (GenBank accession number X53281), ENFDEASKNEAN-COOH, respectively, 9, 14; see also Fig. 34). IF cytosol was not depleted, SRP crosslinked only to signal peptide containing nascent chains (Fig. 3a, lanes 3, 13). To exclude the possibility that our observation resulted from using a heterologous system (Wwheat-germ ribosomes and canine SRP), wwe repeated these experiments in the rabbit reticulocyte lysate translation syste (Fig. 3h). Ai SRP can be crosslinked to all the eytosoldepleted nascent chains ‘When bound to ribosomes alone, SRP can be extracted with high salt”. In contrast, when bound to a signal peptide in the context of the ribosome, SRP is resistant to salt extraction’, Because erosstinking SRP to non-signal peptides may measure proximity rather than binding, we reasoned that if SRP is truly bound to a non-signal peptide, its binding should be resistant to high salt. The experiment in Fig, 3¢ shows this 10 be the ease. Nascent chains of 86aapPL and 77aa‘TLue were assembled and cytosol was removed (Fig. 3c, ‘1x’. After incubation with SRP, dan aliquot of each reaction was irradiated. The remainder of the ‘sample was extracted with high salt a second time and the bound SRP recovered by sedimentation of nasvent-chain complexes. Becauise the amount of SRP that could be crosslinked to the nascent chain was not diminished, we conclude that SRP does interact with noo-signal peptides and is not merely crosslinked NATURE - VOL 370 - 11 AUGUST 1994