The ribosome quality control pathway can access nascent polypeptides stalled at the Sec61 translocon

Cytosolic ribosomes that stall during translation are split into subunits, and nascent polypeptides trapped in the 60S subunit are ubiquitinated by the ribosome quality control (RQC) pathway. Whether the RQC pathway can also target stalls during cotranslational translocation into the ER is not known. Here we report that listerin and NEMF, core RQC components, are bound to translocon-engaged 60S subunits on native ER membranes. RQC recruitment to the ER in cultured cells is stimulated by translation stalling. Biochemical analyses demonstrated that translocon-targeted nascent polypeptides that subsequently stall are polyubiquitinated in 60S complexes. Ubiquitination at the translocon requires cytosolic exposure of the polypeptide at the ribosome–Sec61 junction. This exposure can result from either failed insertion into the Sec61 channel or partial backsliding of translocating nascent chains. Only Sec61-engaged nascent chains early in their biogenesis were relatively refractory to ubiquitination. Modeling based on recent 60S–RQC and 80S–Sec61 structures suggests that the E3 ligase listerin accesses nascent polypeptides via a gap in the ribosome–translocon junction near the Sec61 lateral gate. Thus the RQC pathway can target stalled translocation intermediates for degradation from the Sec61 channel.     

Exploring the capacity of trigger factor to function as a shield for ribosome bound polypeptide chains

Ribosome-bound trigger factor (TF) is the first chaperone encountered by a nascent polypeptide chain in bacteria. TF has been proposed to form a cradle-shaped shield for nascent chains up to approximately 130 residues to fold in a protected environment upon exit from the ribosome. We report that nascent chains of luciferase up to 280 residues in length are relatively protected by TF against digestion by proteinase K. In contrast, nascent chains of the constitutively unstructured protein alpha-synuclein were not protected, although they were in close proximity to TF by crosslinking. Thus, TF is not a general shield for nascent chains. Protease protection appears to depend on a hydrophobic interaction of TF with nascent polypeptides.     

Recognition of nascent polypeptides for targeting and folding

A major difference between the refolding of proteins in vitro and the in vivo folding process, in which we include localization and assembly, is the need for additional factors in vivo, apart from the protein product itself. Thus, the amino acid sequence of a naturally selected protein contains not only the information specifying its three-dimensional structure, but also the information that enables these factors to recognize the nascent polypeptide. In this review, we consider how this latter information may be encoded and, in turn, interpreted by binding species.     

The yeast N(alpha)-acetyltransferase NatA is quantitatively anchored to the ribosome and interacts with nascent polypeptides

The majority of cytosolic proteins in eukaryotes contain a covalently linked acetyl moiety at their very N terminus. The mechanism by which the acetyl moiety is efficiently transferred to a large variety of nascent polypeptides is currently only poorly understood. Yeast N(alpha)-acetyltransferase NatA, consisting of the known subunits Nat1p and the catalytically active Ard1p, recognizes a wide range of sequences and is thought to act cotranslationally. We found that NatA was quantitatively bound to ribosomes via Nat1p and contained a previously unrecognized third subunit, the N(alpha)-acetyltransferase homologue Nat5p. Nat1p not only anchored Ard1p and Nat5p to the ribosome but also was in close proximity to nascent polypeptides, independent of whether they were substrates for N(alpha)-acetylation or not. Besides Nat1p, NAC (nascent polypeptide-associated complex) and the Hsp70 homologue Ssb1/2p interact with a variety of nascent polypeptides on the yeast ribosome. A direct comparison revealed that Nat1p required longer nascent polypeptides for interaction than NAC and Ssb1/2p. Delta nat1 or Delta ard1 deletion strains were temperature sensitive and showed derepression of silent mating type loci while Delta nat5 did not display any obvious phenotype. Temperature sensitivity and derepression of silent mating type loci caused by Delta nat1 or Delta ard1 were partially suppressed by overexpression of SSB1. The combination of data suggests that Nat1p presents the N termini of nascent polypeptides for acetylation and might serve additional roles during protein synthesis.     

Trigger factor binding to ribosomes with nascent peptide chains of varying lengths and sequences

Trigger factor (TF) is the first protein-folding chaperone to interact with a nascent peptide chain as it emerges from the ribosome. Here, we have used a spin down assay to estimate the affinities for the binding of TF to ribosome nascent chain complexes (RNCs) with peptides of varying lengths and sequences. An in vitro system for protein synthesis assembled from purified Escherichia coli components was used to produce RNCs stalled on truncated mRNAs. The affinity of TF to RNCs exposing RNA polymerase sequences increased with the length of the nascent peptides. TF bound to RNA polymerase RNCs with significantly higher affinity than to inner membrane protein leader peptidase and bacterioopsin RNCs. The latter two RNCs are substrates for signal recognition particle, suggesting complementary affinities of TF and signal recognition particle to nascent peptides targeted for cytoplasm and membrane.     

Delivery of nascent polypeptides to the mitochondrial surface

Thousands of polypeptides with diverse biochemical properties, some of which are extremely hydrophobic, are targeted from cytoplasmic ribosomes to the surface of mitochondria. Localised synthesis, as well as transient interactions with a wide array of molecular chaperones and other cytoplasmic factors, can promote productive interaction of mitochondrial proteins with the TOM complex to initiate protein import into mitochondria.     

Translation arrest requires two-way communication between a nascent polypeptide and the ribosome

When the export of E. coli SecM is blocked, a 17 amino acid motif near the C terminus of the protein induces a translation arrest from within the ribosome tunnel. Here we used a recently described application of fluorescence resonance energy transfer (FRET) to gain insight into the mechanism of translation arrest. We found that the SecM C terminus adopted a compact conformation upon synthesis of the arrest motif. This conformational change did not occur spontaneously, but rather was induced by the ribosome. Translation arrest required both compaction of the SecM C terminus and the presence of key residues in the arrest motif. Further analysis showed that the arrested peptidyl-tRNA was resistant to puromycin treatment and revealed additional changes in the ribosome-nascent SecM complex. Based on these observations, we propose that translation arrest results from a series of reciprocal interactions between the ribosome and the C terminus of the nascent SecM polypeptide.     

Interplay of signal recognition particle and trigger factor at L23 near the nascent chain exit site on the Escherichia coli ribosome

As newly synthesized polypeptides emerge from the ribosome, they interact with chaperones and targeting factors that assist in folding and targeting to the proper location in the cell. In Escherichia coli, the chaperone trigger factor (TF) binds to nascent polypeptides early in biosynthesis facilitated by its affinity for the ribosomal proteins L23 and L29 that are situated around the nascent chain exit site on the ribosome. The targeting factor signal recognition particle (SRP) interacts specifically with the signal anchor (SA) sequence in nascent inner membrane proteins (IMPs). Here, we have used photocross-linking to map interactions of the SA sequence in a short, in vitro-synthesized, nascent IMP. Both TF and SRP were found to interact with the SA with partially overlapping binding specificity. In addition, extensive contacts with L23 and L29 were detected. Both purified TF and SRP could be cross-linked to L23 on nontranslating ribosomes with a competitive advantage for SRP. The results suggest a role for L23 in the targeting of IMPs as an attachment site for TF and SRP that is close to the emerging nascent chain.     

A cradle for new proteins: trigger factor at the ribosome

Newly synthesized proteins leave the ribosome through a narrow tunnel in the large subunit. During ongoing synthesis, nascent protein chains are particularly sensitive to aggregation and degradation because they emerge from the ribosome in an unfolded state. In bacteria, the first protein to interact with nascent chains and facilitate their folding is the ribosome-associated chaperone trigger factor. Recently, crystal structures of trigger factor and of its ribosome-binding domain in complex with the large ribosomal subunit revealed that the chaperone adopts an extended 'dragon-shaped' fold with a large hydrophobic cradle, which arches over the exit of the ribosomal tunnel and shields newly synthesized proteins. These structural results, together with recent biochemical data on trigger factor and its interplay with other chaperones and factors that interact with the nascent chain, provide a comprehensive view of the role of trigger factor during co-translational protein folding.     

Sequence-specific interactions of nascent Escherichia coli polypeptides with trigger factor and signal recognition particle

As nascent polypeptides exit the ribosomal tunnel they immediately associate with chaperones, folding catalysts, and targeting factors. These interactions are decisive for the future conformation and destination of the protein that is being synthesized. Using Escherichia coli as a model organism, we have systematically analyzed how the earliest contacts of nascent polypeptides with cytosolic factors depend on the nature and future destination of the emerging sequence using a photo cross-linking approach. Together, the data suggest that the chaperone trigger factor is adjacent to emerging sequences by default, consistent with both its placement near the nascent chain exit site and its cellular abundance. The signal recognition particle (SRP) effectively competes the contact with TF when a signal anchor (SA) sequence of a nascent inner membrane protein appears outside the ribosome. The SRP remains in contact with the SA and downstream sequences during further synthesis of approximately 30 amino acids. The contact with trigger factor is then restored unless another transmembrane segment reinitiates SRP binding. Importantly and in contrast to published data, the SRP appears perfectly capable of distinguishing SA sequences from signal sequences in secretory proteins at this early stage in biogenesis.     

Generation of ribosome nascent chain complexes for structural and functional studies

Biochemical and structural studies of co-translational folding, targeting and translocation depend on an efficient methodology to prepare ribosome nascent chain complexes (RNCs). Here we present our approach for the generation of homogenous and stable RNCs involving in vitro translation and affinity purification. Fusing the SecM arrest sequence, which tightly interacts with the ribosomal tunnel, to the nascent polypeptide chain significantly enhanced the stability of the RNCs. We have been able to increase the yield of the affinity purification step by engineering a tag with higher affinity. The RNCs generated with this approach have been successfully used to obtain 3D cryo-electron microscopic reconstructions of complexes with the signal recognition particle and the translocon. The established procedure is highly efficient and if scaled up could yield milligram amounts of RNCs sufficient for crystallization experiments.     

Trigger factor interacts with the signal peptide of nascent Tat substrates but does not play a critical role in Tat-mediated export.

Twin-arginine translocation (Tat)-mediated protein transport across the bacterial cytoplasmic membrane occurs only after synthesis and folding of the substrate protein that contains a signal peptide with a characteristic twin-arginine motif. This implies that premature contact between the Tat signal peptide and the Tat translocon in the membrane must be prevented. We used site-specific photo-crosslinking to demonstrate that the signal peptide of nascent Tat proteins is in close proximity to the chaperone and peptidyl-prolyl isomerase trigger factor (TF). The contact with TF was strictly dependent on the context of the translating ribosome, started early in biogenesis when the nascent chain left the ribosome near L23, and persisted until the chain reached its full length. Despite this exclusive and prolonged contact, depletion or overexpression of TF had little effect on the kinetics and efficiency of the Tat export process.     

Elongation factor G participates in ribosome disassembly by interacting with ribosome recycling factor at their tRNA-mimicry domains

Elongation factor G (EF-G) is a G protein with motor function that drives two target molecules, a tRNA in the translating ribosome and the ribosome recycling factor (RRF) in the post-termination complex. How G protein motor action is transmitted to RRF is unknown. Thermus thermophilus RRF is nonfunctional in Escherichia coli. It became functional upon introducing a plasmid expressing E. coli EF-G with surface changes in its tRNA-mimic domain or by replacing the E. coli EF-G tRNA-mimic domain by the Thermus domain. Thermus RRF could also be activated by introducing surface substitutions in its anticodon arm-mimic region. These gain-of-function phenotypes depend on the combination of heterologous EF-G and RRF alleles. These mutational studies suggest that EF-G motor action is transmitted to RRF by specific surface contacts between the domains that mimic the anticodon arm.     

Different conformations of nascent polypeptides during translocation across the ER membrane

Background  

In eukaryotic cells, proteins are translocated across the ER membrane through a continuous ribosome-translocon channel. It is unclear to what extent proteins can fold already within the ribosome-translocon channel, and previous studies suggest that only a limited degree of folding (such as the formation of isolated α-helices) may be possible within the ribosome.     

Studies on the accessibility of nascent non-helical peptide chains on the ribosome

The accessibility of nascent polypeptides with special structural elements to the ribosome was investigated. Poly(C), poly(C, U) and poly(C, A) mRNAs were translated by E. coli ribosomes in vitro. The resulting peptides which were rich in prolines, remained on the ribosomal particles or were released after addition of puromycin. A protease from Aspergillus oryzae hydrolyzed the released peptides rapidly, whereas the degradation of the unreleased ones was only slightly affected. This result shows that the nascent peptides were protected against proteolytic attack by the ribosomal particles. Interestingly, the protease completely degraded the 30S particles whereas the 50S ones remained intact, even after prolonged incubation.     

The oxidative folding of nascent polypeptides provides electrons for reductive reactions in the ER

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