Dual binding mode of the Nascent Polypeptide-associated Complex (NAC) reveals a novel universal adapter site on the ribosome

Nascent Polypeptide-associated Complex (NAC) was identified in eukaryotes as the first cytosolic factor which contacts the nascent polypeptide chain emerging from the ribosome. NAC is present as a homodimer in Archaea and as a highly conserved heterodimer in eukaryotes. Mutations in NAC cause severe embryonically lethal phenotypes in mice, D. melanogaster and C. elegans. In the yeast Saccharomyces cerevisiae NAC is quantitatively associated with ribosomes. Here we show that NAC contacts several ribosomal proteins. The N-terminus of βNAC, however, specifically contacts near the tunnel exit the ribosomal protein Rpl31, which is unique to Eukaryotes and Archaea. Moreover, the first 23 amino acids of βNAC are sufficient to direct an otherwise nonassociated protein to the ribosome. In contrast, αNAC (Egd2p) contacts Rpl17, the direct neighbour of Rpl31 at the ribosomal tunnel exit site. Rpl31 was recently also identified as a contact site for the SRP receptor and the ribosome associated complex (RAC). Furthermore, in E. coli peptide deformylase (PDF) interacts with the corresponding surface area on the eubacterial ribosome. In addition to the previously identified universal adapter site represented by Rpl25/35, we therefore refer to Rpl31/Rpl17 as a novel universal docking site for ribosome-associated factors on the eukaryotic ribosome. Introduction The biosynthesis of proteins by ribosomes is an essential process in all living cells. As soon as a newly synthesized polypeptide emerges from the ribosomal exit tunnel several ribosomeassociated factors, which are involved in maturation, folding and/or sorting of the protein, contact the nascent chain (1-3). In eubacteria, these associated factors include trigger factor (TF), the modifying enzymes peptidyl deformylase (PDF) and methionine aminopeptidase (MAP), signal recognition particle (SRP) and its receptor (SR), and the translocation pore SecY. In contrast, in eukaryotes a larger variety of such factors must gain access to the nascent chain (the chaperones RAC and SSB, the Nascent Polypeptideassociated Complex (NAC), various MAPs and N-acetyl transferases, the ribosome-associated membrane protein ERj1p as well as SRP, SR and the translocon Sec61. Since the available interaction space around the ribosomal tunnel exit is rather limited, these factors must have developed different strategies to encounter the nascent chain. The ribosomal exit tunnel, which itself is mainly formed by ribosomal RNA, is surrounded at its exit site by a small set of ribosomal proteins. Whereas some of these are universally conserved through all domains of life (Rpl17, Rpl25, Rpl26 and Rpl35 in yeast corresponding to L22, L23, L24 and L29 in eubacteria), others are either restricted to eubacteria (L17 and L32) or only found in archaea and eukaryotes (Rpl19, Rpl31 and Rpl39) (4-7). Based on crosslinking experiments and structural studies Rpl25/Rpl35 (L23/L29 in eubacteria) have been identified so far as a general docking site for the ribosome associated factors trigger factor, SRP, the translocon Sec61 or SecY, the ribosome-associated membrane protein ERj1p and the “insertases” YidC or Oxa1, respectively (8-13). In eukaryotes, NAC was identified as the first ribosome-associated factor to contact the emerging polypeptide chain (14). NAC is a highly abundant heterodimeric cytosolic protein complex composed of αNAC and βNAC, which show substantial homology to each other (15). 1 http://www.jbc.org/cgi/doi/10.1074/jbc.M109.092536 The latest version is at JBC Papers in Press. Published on April 21, 2010 as Manuscript M109.092536 Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from NAC binds to ribosome at novel universal adapter site Pech et al. Although not essential in S. cerevisiae, the importance of NACs in vivo function is emphasized by early embryonically lethal phenotypes of NAC mutants in higher eukaryotes, such as Mus musculus, Drosophila melanogaster and Caenorhabditis elegans (1618). Intracellular levels of the individual NAC subunits change in the context of several human diseases, such as Alzheimer ́s disease, Down Syndrom, AIDS, malignant brain tumors, and a role for NAC in apoptosis was proposed (16,1923). NAC does not show similarity to other proteins and its cellular function is still poorly understood. Early protease protection assays suggested that NAC might function as a shield for newly synthesized polypeptides on the ribosome against inappropriate interaction with cytosolic factors (24). Cycles of binding and releasing NAC, were proposed to expose the polypeptide to the cytosol in “quantal units”, rather than amino acid by amino acid. Thereby NAC would contribute to fidelity in cotranslational processes such as targeting and folding (25). Additional studies suggested that NAC is involved in regulation of ribosome access to the translocation pore in the ER membrane in cotranslational protein translocation (26-28). Yeast NAC was shown to play a role in the attachment of cytosolic ribosomes to mitochondria (29,30) and in translation-coupled import of proteins into mitochondria (31,32). For the human αNAC also transcription related functions have been described (33,34). Both NAC subunits seem to differ with respect to their function. While both subunits contact the nascent chain on the ribosome, as was shown by crosslinking, βNAC alone was sufficient for binding to the ribosome and prevention of ribosome interaction with the translocon (15,35). In contrast, only the heterodimeric complex prevents inappropriate interaction with the nascent chain (15). The availability of numerous completed archaebacterial genomes revealed that all archaeal taxa contain one gene with apparent homology to αNAC (36). The crystal structure of archaeal NAC (aeNAC) uncovered that it forms a homodimer exhibiting two folded domains (37). A ubiquitin-associated domain (UBA domain) at the C-terminus of aeNAC, which is also found in all eukaryotic αNAC proteins, and the central NAC domain providing the dimerization interface. The central NAC domain exhibits a unique novel protein fold. It resembles a flattened beta barrel which exposes several hydrophobic residues on one of its concave surfaces. This domain is structurally conserved from archaea to humans emphasizing its importance for NACs function. The observation of crosslinks between NAC and very short nascent chains (24) imply that NACs binding site on the ribosome must be in close proximity to the tunnel exit on the large ribosomal subunit. Recently, Rpl25 was suggested as NACs binding site on the ribosome based on a heterologous crosslinking assay using yeast NAC and E. coli ribosomes (38). In order to further characterize NACs function we wanted to investigate its interaction with the ribosome in more detail. Therefore we followed a crosslinking approach to identify the major binding site of NAC on the ribosome. Based on these results we constructed various NAC mutants and studied the sedimentation behaviour of these NAC variants. In this report we show that NAC interacts with several ribosomal proteins. The pivotal contact is made to Rpl31 via the N-terminus of βNAC. We show that the ability to interact with ribosomes can be conferred by fusing this N-terminus to a protein which is otherwise not associated with ribosomes. In addition, we show that αNAC contacts Rpl17, the direct neighbour of Rpl31 on the ribosomal surface and propose a model for NACs interaction with this novel universal docking site at the ribosomal tunnel exit. Experimental Procedures: Materials: Chemicals and secondary antibodies were purchased from Merck and Sigma, restriction enzymes and Vent polymerase from New England Biolabs, protease inhibitor cocktail was from Roche, RNase inhibitor from Promega. Crosslinking reagents were purchased from Pierce and Molecular Biosciences. Samples were analyzed on precast Bis-Tris gels from Invitrogen. Strains: S. cerevisiae strain W303-1A and the NAC knock out strain YJF24 (Δegd2; Δegd1; Δbtt1) were a gift of B. Wiedmann (35). Strain MH272-3fα and the ΔRpl31A/B strain were provided by S. Rospert (39). Cloning was performed in E. coli XL1blue (Stratagene), expression in E. coli ER2566 (New England Biolabs). Cloning, expression and purification: Cloning experiments were performed following standard protocols (40).The genes egd2 and egd1 were 2 by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from NAC binds to ribosome at novel universal adapter site Pech et al. amplified from yeast genomic DNA and cloned together into pET28a (Novagen) leading to a His-tag fusion followed by a thrombin cleavage site at the N-terminus of αNAC. The genes were expressed in E. coli ER2566 and the protein complex purified via Ni-NTA agarose (Qiagen, according to the manufacturers manual) followed by heparin affinity chromatography (POROS 20 HE, Applied Biosystems) and anion exchange chromatography (POROS 20 HQ, Applied Biosystems). The His-tag was removed from αNAC by thrombin cleavage during the purification procedure. In addition, egd1 and its alanine substitution mutants were cloned into pRS425-GAL and egd2 into pYES2.1 (Invitrogen) for expression in YJF24. Antibodies and immunoblotting procedures: The genes coding for yeast Rpl4, Rpl35, and Rpl25 were cloned into pET28a, expressed in E. coli, purified via a His-tag and used for immunization of chicken (David Biotechnologies, Regensburg). Preimmune sera did not react with any protein in a Western blot with total cell extract from S. cerevisiae prepared according to Yaffe and Schatz (41). Proteins were separated on 10 % NuPAGE BisTris gels using MES buffer (Invitrogen) followed by transfer onto nitrocellulose or PVDF membrane using Bis Bicine transfer buffer (25mM Bicine, 25 mM Bis-Tris, 1 mM EDTA free acid, 20 % methanol). Secondary antibodies were conjugated with Horse Radish Peroxidase and detected using the ECL kit (Amersham). Site specific mutagenesis: Site specific introduction of unique cysteine residues in the N-terminus of βNAC at positions 3,5,7,9 or 11 as well as replacement of EKL and KLQ against AAA in βNAC were performed following the “Quick change site directed mutagenesis” protocol (Stratagene). Fusion genes coding for the first 14, 23 or 39 Nterminal amino acids of βNAC fused via a (Gly4, Ser)3 linker to the full length gene of maltose binding protein from E. coli were constructed using standard PCR techniques, cloned into pYES2.1 and expressed in YJF24. Isolation of ribosomes from Yeast: Yeast cultures were grown to an OD600 of 1.5 on YPD medium, cells were collected, washed with ice cold ddH2O and 1% KCl, followed by an incubation with 100 mM TRIS-HCl pH 8.0, 10 mM DTT for 15 min at 30°C. Cells were collected, resuspended in lysis buffer (20 mM Hepes-KOH pH 7.5, 100 mM KOAc, 5 mM Mg(OAc)2, 4 mM DTT, 150 mM sucrose, 500 μM PMSF, protease inhibitor) and disrupted by three passages through an EmulsiFlex-C5 High Pressure homogenizer at 1.400 bar. Cell debris was separated by spinning for 15 min in a SS34 rotor at 15.500 rpm. The supernatant was spun again in a Ti 60 rotor for 30 min at 35.700 rpm. The resulting supernatant was termed S100. Ribosomes were pelleted from this S100 extract through a highsalt (500 mM KOAc) or low salt (100 mM KOAc) sucrose cushion (20 mM Hepes-KOH pH 7.5, 100-500 mM KOAc, 5 mM Mg(OAc)2, 2 mM DTT, protease inhibitor, 1 M sucrose) in a TLA 100.3 rotor for 1 h at 100.000 rpm. Chemical cross-linking of NAC and ribosomal proteins using AMAS: A twofold molar excess of yeast NAC was incubated with high salt stripped ribosomes isolated from the NAC knock out yeast strain YJF24 as follows: 80S ribosomes (4 μM) were incubated with 8 μM recombinant NAC (in 20 mM Hepes pH 7.5, 150 mM KOAc, 20 mM Mg(OAc)2, protease inhibitor cocktail complete) for 2 min at 26°C followed by 5 min on ice. AMAS (N-[αmaleimidoacetoxy] succinimide ester) in DMSO was added to a final concentration of 1.6 mM. After 2 h at 4°C the reaction was stopped by a 50 fold excess of glycine and incubation at 25°C for 30 min. Reactions were brought to high salt by addition of KOAc to 0.5 M, incubated on ice for 30 min before ribosome bound material was pelleted through a 0.5 M sucrose cushion (20 mM Hepes pH 7.5, 500 mM KOAc, 20 mM Mg(OAc)2, protease inhibitor cocktail complete) at 355.000 x g in a TLA-100.2 rotor. Pellets were resuspended in RNase buffer (50 mM Tris-HCl pH 8.0, 0.5 M NaCl, 2 mM EDTA, 0.5 mg/ml RNaseA, protease inhibitor cocktail complete), incubated for 1 h at 37°C before addition of SDS sample buffer and separation on 10 % Bis-Tris gels (Invitrogen). Site-specific cross-linking of NAC and ribosomal proteins using BPIA: Site specific cross linking with recombinant NAC carrying unique cysteins at the N-terminus of βNAC was performed in principle according to (42). 6 μM NAC (in 20 mM Hepes-KOH pH 7.5; 150 mM KOAc, 20 mM Mg(OAc)2; 5 mM TCEP (Tris(2-carboxyethyl)-phosphin); protease inhibitor complete) were incubated for 30 min at 30°C to 3 by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from NAC binds to ribosome at novel universal adapter site Pech et al. reduce possibly formed disulfide bridges. BPIA (Benzophenone-4-iodoacetamide) in dimethyl formamide was added to a final concentration of 12 μM followed by a 1 h incubation at RT in the dark. DTT was added in a fivefold excess and incubation was continued for 15 min. High salt purified 80S ribosomes from YJF24 were added to a final concentration of 3 μM, resulting in a two molar excess on NAC over ribosomes. Samples were incubated for 2 min at 26°C and 5 min on ice in the dark. The crosslinker was activated by irradiation with UV light (Blak Ray lamp model B100 AP 100W 365 nm for 5 min at 5 cm distance). The samples were then high salt treated and ribosome bound material was treated as described above. Analyses of NACs association with ribosomes: Ribosomes were isolated under low salt (100 mM KOAc) conditions. Yeast cultures (W-3031A or YJF24 expressing egd1 or egd2 from plasmids) were grown on YPD medium or the appropriate selection medium to an OD600 of 1.0 to 1.5. Cells were harvested (7.000 x g; 6 min; 4°C), washed with ice cold ddH2O followed by 1% KCl. Cells were then incubated in 100 mM Tris-HCl pH 8.0; 10 mM DTT for 15 min at 30°C, pelleted again, resuspended in lysis buffer (20 mM Hepes-KOH pH 7.5; 100 mM KOAc, 20 mM Mg(OAc)2, 4 mM DTT, 150 mM sucrose, 500 μM PMSF, protease inhibitor cocktail complete) and disrupted by 3 passages through an EmulsiFlex-C5 High Pressure homogenizer at 1.400 bar. Cell debris was separated at 16.000 x g for 15 min. The supernatant was spun again at 100.000 x g for 30 min to yield the S100 extract which was quick frozen in liquid nitrogen or directly used for sedimentation of ribosomes. 1 ml sucrose cushion (20 mM Hepes-KOH pH 7.5; 100 mM KOAc, 20 mM Mg(OAc)2, 4 mM DTT, 1 M sucrose, 500 μM PMSF, protease inhibitor cocktail complete) was overlayed with 2 ml S100 and ribosomes were pelleted for 1 h at 540.000 x g and 4°C. The supernatant was carefully removed and ribosomes were resuspended in ribosome buffer (20 mM HepesKOH pH 7.5; 100 mM KOAc, 20 mM Mg(OAc)2, 1 mM DTT, 500 μM PMSF, protease inhibitor cocktail complete). Equivalent amounts of ribosomes and supernatant were analysed by immunoblotting. In vitro binding of NAC variants to ribosomes: High salt stripped ribosomes (4 μM) from yeast (YJF24) or E. coli (W3110) were incubated with a two molar excess of recombinant NAC (8 μM in 20 mM Hepes-KOH pH 7.5, 150 mM KOAc, 20 mM Mg(OAc)2 , 1 mM DTT, protease inhibitor) for 2 min at 26°C followed by 5 min on ice. Ribosomes were sedimented through a 1 M sucrose cushion (20 mM Hepes-KOH pH 7.5, 100 mM KOAc, 20 mM Mg(OAc)2 , 1 mM DTT, 1 M sucrose, protease inhibitor) for 1 h at 355.000 x g at 4°C. Equivalent amounts of the ribosomes and the supernatant were analyzed by immunoblotting.