Nuclear translation visualized by ribosome-bound nascent chain puromycylation

Whether protein translation occurs in the nucleus is contentious. To address this question, we developed the ribopuromycylation method (RPM), which visualizes translation in cells via standard immunofluorescence microscopy. The RPM is based on ribosome-catalyzed puromycylation of nascent chains immobilized on ribosomes by antibiotic chain elongation inhibitors followed by detection of puromycylated ribosome-bound nascent chains with a puromycin (PMY)-specific monoclonal antibody in fixed and permeabilized cells. The RPM correlates localized translation with myriad processes in cells and can be applied to any cell whose translation is sensitive to PMY. In this paper, we use the RPM to provide evidence for translation in the nucleoplasm and nucleolus, which is regulated by infectious and chemical stress.     

The molecular chaperone Ssb from Saccharomyces cerevisiae is a component of the ribosome-nascent chain complex.

The 70 kDa heat shock proteins (Hsp70s) are a ubiquitous class of molecular chaperones. The Ssbs of Saccharomyces cerevisiae are an abundant type of Hsp70 found associated with translating ribosomes. To understand better the function of Ssb in association with ribosomes, the Ssb-ribosome interaction was characterized. Incorporation of the aminoacyl-tRNA analog puromycin by translating ribosomes caused the release of Ssb concomitant with the release of nascent chains. In addition, Ssb could be cross-linked to nascent chains containing a modified lysine residue with a photoactivatable cross-linker. Together, these results suggest an interaction of Ssb with the nascent chain. The interaction of Ssb with the ribosome-nascent chain complex was stable, as demonstrated by resistance to treatment with high salt; however, Ssb interaction with the ribosome in the absence of nascent chain was salt sensitive. We propose that Ssb is a core component of the translating ribosome which interacts with both the nascent polypeptide chain and the ribosome. These interactions allow Ssb to function as a chaperone on the ribosome, preventing the misfolding of newly synthesized proteins.     

Homogeneous stalled ribosome nascent chain complexes produced in vivo or in vitro

Cotranslational protein maturation is often studied in cell-free translation mixtures, using stalled ribosome-nascent chain complexes produced by translating truncated mRNA. This approach has two limitations: (i) it can be technically challenging, and (ii) it only works in vitro, where the concentrations of cellular components differ from concentrations in vivo. We have developed a method to produce stalled ribosomes bearing nascent chains of a specified length by using a 'stall sequence', derived from the Escherichia coli SecM protein, which interacts with residues in the ribosomal exit tunnel to stall SecM translation. When the stall sequence is expressed at the end of nascent chains, stable translation-arrested ribosome complexes accumulate in intact cells or cell-free extracts. SecM-directed stalling is efficient, with negligible effects on viability. This method is straightforward and suitable for producing stalled ribosome complexes in vivo, permitting study of the length-dependent maturation of nascent chains in the cellular milieu.     

Genetic depletion of the RNA helicase DDX3 leads to impaired elongation of translating ribosomes triggering co-translational quality control of newly synthesized polypeptides

DDX3 is a multifaceted RNA helicase of the DEAD-box family that plays central roles in all aspects of RNA metabolism including translation initiation. Here, we provide evidence that the Leishmania DDX3 ortholog functions in post-initiation steps of translation. We show that genetic depletion of DDX3 slows down ribosome movement resulting in elongation-stalled ribosomes, impaired translation elongation and decreased de novo protein synthesis. We also demonstrate that the essential ribosome recycling factor Rli1/ABCE1 and termination factors eRF3 and GTPBP1 are less recruited to ribosomes upon DDX3 loss, suggesting that arrested ribosomes may be inefficiently dissociated and recycled. Furthermore, we show that prolonged ribosome stalling triggers co-translational ubiquitination of nascent polypeptide chains and a higher recruitment of E3 ubiquitin ligases and proteasome components to ribosomes of DDX3 knockout cells, which further supports that ribosomes are not elongating optimally. Impaired elongation of translating ribosomes also results in the accumulation of cytoplasmic protein aggregates, which implies that defects in translation overwhelm the normal quality controls. The partial recovery of translation by overexpressing Hsp70 supports this possibility. Collectively, these results suggest an important novel contribution of DDX3 to optimal elongation of translating ribosomes by preventing prolonged translation stalls and stimulating recycling of arrested ribosomes.     

Association of protein biogenesis factors at the yeast ribosomal tunnel exit is affected by the translational status and nascent polypeptide sequence

Ribosome-associated protein biogenesis factors (RPBs) act during a short but critical period of protein biogenesis. The action of RPBs starts as soon as a nascent polypeptide becomes accessible from the outside of the ribosome and ends upon termination of translation. In yeast, RPBs include the chaperones Ssb1/2 and ribosome-associated complex, signal recognition particle, nascent polypeptide-associated complex (NAC), the aminopeptidases Map1 and Map2, and the Nalpha-terminal acetyltransferase NatA. Here, we provide the first comprehensive analysis of RPB binding at the yeast ribosomal tunnel exit as a function of translational status and polypeptide sequence. We measured the ratios of RPBs to ribosomes in yeast cells and determined RPB occupation of translating and non-translating ribosomes. The combined results imply a requirement for dynamic and coordinated interactions at the tunnel exit. Exclusively, NAC was associated with the majority of ribosomes regardless of their translational status. All other RPBs occupied only ribosomal subpopulations, binding with increased apparent affinity to randomly translating ribosomes as compared with non-translating ones. Analysis of RPB interaction with homogenous ribosome populations engaged in the translation of specific nascent polypeptides revealed that the affinities of Ssb1/2, NAC, and, as expected, signal recognition particle, were influenced by the amino acid sequence of the nascent polypeptide. Complementary cross-linking data suggest that not only affinity of RPBs to the ribosome but also positioning can be influenced in a nascent polypeptide-dependent manner.     

Saccharomyces cerevisiae translational activator Cbs1p is associated with translationally active mitochondrial ribosomes

In the yeast Saccharomyces cerevisiae, mitochondrial translation of most, if not all, mitochondrially encoded genes is regulated by an individual set of gene-specific activators. Translation of the COB mRNA encoding cytochrome b requires the function of two nuclearly encoded proteins, Cbs1p and Cbs2p. Genetic data revealed that the 5'-untranslated region of COB mRNA is the target of both proteins. Recently, we provided evidence for an interaction of Cbs2p with mitochondrial ribosomes. We demonstrate here by means of blue native gel electrophoresis, density gradient centrifugation and tandem affinity purification that a portion of Cbs1p is also associated with mitochondrial ribosomes. In addition, we demonstrate that the amount of ribosome-associated Cbs1p is elevated in the presence of chloramphenicol, which is known to stall ribosomes on mRNAs. In the presence of puromycin, which strips off the mRNA and nascent protein chains from ribosomes, Cbs1p is no longer associated with ribosomes. Our data indicate that the observed interaction is mediated by ribosome-bound mRNA, thus restricting the association to ribosomes actively translating cytochrome b.     

The translation machinery and 70 kd heat shock protein cooperate in protein synthesis.

The function of the yeast SSB 70 kd heatshock proteins (hsp70s) was investigated by a variety of approaches. The SSB hsp70s (Ssb1/2p) are associated with translating ribosomes. This association is disrupted by puromycin, suggesting that Ssb1/2p may bind directly to the nascent polypeptide. Mutant ssb1 ssb2 strains grow slowly, contain a low number of translating ribosomes, and are hypersensitive to several inhibitors of protein synthesis. The slow growth phenotype of ssb1 ssb2 mutants is suppressed by increased copy number of a gene encoding a novel translation elongation factor 1 alpha (EF-1 alpha)-like protein. We suggest that cytosolic hsp70 aids in the passage of the nascent polypeptide chain through the ribosome in a manner analogous to the role played by organelle-localized hsp70 in the transport of proteins across membranes.     

Immunological studies of the fragments of bovine cartilage proteoglycan produced by chondroitinase-trypsin digestion.

The peptidyl transferase activity of polysomes from Escherichia coli, rabbit reticulocytes and chick embryos, assayed in the fragment reaction, is 3- to 10-fold lower than the corresponding activity of single ribosomes. The polysomal peptidyl transferase activity is restored in full under conditions of in vitro protein synthesis that result in conversion of polysomes to single ribosomes. Thus, the peptidyl transferase center is masked in translating ribosomes. Unmasking of peptidyl transferase, however, does not require the release of ribosomes from messenger RNA: it is also seen upon treatment of polysomes with puromycin, under conditions in which polysomes remain intact. Apparently, release of nascent polypeptide chains is sufficient to allow access of formylmethionyl hexanucleotide substrate to the peptidyl transferase site.     

RIBOSOMES BOUND TO CHLOROPLAST MEMBRANES IN CHLAMYDOMONAS REINHARDTII

The amount of chloroplast ribosomal RNAs of Chlamydomonas reinhardtii which sediment at 15,000 g is increased when cells are treated with chloramphenicol. Preparations of chloroplast membranes from chloramphenicol-treated cells contain more chloroplast ribosomal RNAs than preparations from untreated cells. The membranes from treated cells also contain more ribosome-like particles, some of which appear in polysome-like arrangements. About 50% of chloroplast ribosomes are released from membranes in vitro as subunits by 1 mM puromycin in 500 mM KCl. A portion of chloroplast ribosomal subunits is released by 500 mM KCl alone, a portion by 1 mM puromycin alone, and a portion by 1 mM puromycin in 500 mM KCl. Ribosomes are not released from isolated membranes by treatment with ribonuclease. Membranes in chloroplasts of chloramphenicol-treated cells show many ribosomes associated with membranes, some of which are present in polysome-like arrangements. This type of organization is less frequent in chloroplasts of untreated cells. Streptogramin, an inhibitor of initiation, prevents chloramphenicol from acting to permit isolation of membrane-bound ribosomes. Membrane-bound chloroplast ribosomes are probably a normal component of actively growing cells. The ability to isolate membrane-bound ribosomes from chloramphenicol-treated cells is probably due to chloramphenicol-prevented completion of nascent chains during harvesting of cells. Since chloroplasts synthesize some of their membrane proteins, and a portion of chloroplast ribosomes is bound to chloroplast membranes through nascent protein chains, it is suggested that the membrane-bound ribosomes are synthesizing membrane protein.     

Selective SecA association with signal sequences in ribosome-bound nascent chains: a potential role for SecA in ribosome targeting to the bacterial membrane

The role of SecA in selecting bacterial proteins for export was examined using a heterologous system that lacks endogenous SecA and other bacterial proteins. This approach allowed us to assess the interaction of SecA with ribosome-bound photoreactive nascent chains in the absence of trigger factor, SecB, Ffh (the bacterial protein component of the signal recognition particle), and the SecYEG translocon in the bacterial plasma membrane. In the absence of membranes, SecA photocross-linked efficiently to nascent translocation substrate OmpA in ribosome-nascent chain (RNC) complexes in an interaction that was independent of both ATP and SecB. However, no photocross-linking to a nascent membrane protein that is normally targeted by a signal recognition particle was observed. Modification of the signal sequence revealed that its affinity for SecA and Ffh varied inversely. Gel filtration showed that SecA binds tightly to both translating and non-translating ribosomes. When purified SecA.RNC complexes containing nascent OmpA were exposed to inner membrane vesicles lacking functional SecA, the nascent chains were successfully targeted to SecYEG translocons. However, purified RNCs lacking SecA were unable to target to the same membranes. Taken together, these data strongly suggest that cytosolic SecA participates in the selection of proteins for export by co-translationally binding to the signal sequences of non-membrane proteins and directing those nascent chains to the translocon.     

A polysome-membrane binding system from rat liver. I. Basic characterization of the binding system.

A simple reaction system was developed to examine the binding of polysomes to membranes of the endoplasmic reticulum and to investigate the fate of ribosomes and nascent chains during protein synthesis in vitro. The system conssited of Sephadex G-25 treated post-mitochondrial fraction prepared from rat liver (Sephadex-PM) as a source of membranes, and radioactive free polysomes prepared from another rat liver. The following results were obtained. 1. Nascent chains on free polysomes labeled in vivo were transferred to membranes in vitro. The process required protein synthesis. 2. This reaction occurred in two steps: a) Binding of the free polysomes to membranes in the absence of protein synthesis. b) Release of ribosomes, leaving nascent chains on the membranes, requiring protein syntehsis. 3. A portion of the ribosomes found on membranes in vivi (membrane-bound ribosomes) was also released from the membranes during incubation in vitro, leaving their nascent chains on the membranes. The significance of the transfer of nascent chains from free polysomes to membranes in vitro is discussed in the light of known polysome-membrane interaction in vivo.     

Signal recognition particle binds to ribosome-bound signal sequences with fluorescence-detected subnanomolar affinity that does not diminish as the nascent chain lengthens

The binding of signal recognition particle (SRP) to ribosome-bound signal sequences has been characterized directly and quantitatively using fluorescence spectroscopy. A fluorescent probe was incorporated cotranslationally into the signal sequence of a ribosome.nascent chain complex (RNC), and upon titration with SRP, a large and saturable increase in fluorescence intensity was observed. Spectral analyses of SRP and RNC association as a function of concentration allowed us to measure, at equilibrium, K(d) values of 0.05-0.38 nm for SRP.RNC complexes with different signal sequences. Competitive binding experiments with nonfluorescent RNC species revealed that the nascent chain probe did not alter SRP affinity and that SRP has significant affinity for both nontranslating ribosomes (K(d) = 71 nm) and RNCs that lack an exposed signal sequence (K(d) = 8 nm). SRP can therefore distinguish between translating and nontranslating ribosomes. The very high signal sequence-dependent SRP.RNC affinity did not decrease as the nascent chain lengthened. Thus, the inhibition of SRP-dependent targeting of RNCs to the endoplasmic reticulum membrane observed with long nascent chains does not result from reduced SRP binding to the signal sequence, as widely thought, but rather from a subsequent step, presumably nascent chain interference of SRP.RNC association with the SRP receptor and/or translocon.     

RIBOSOME-MEMBRANE INTERACTION : Nondestructive Disassembly of Rat Liver Rough Microsomes into Ribosomal and Membranous Components

In a medium of high ionic strength, rat liver rough microsomes can be nondestructively disassembled into ribosomes and stripped membranes if nascent polypeptides are discharged from the bound ribosomes by reaction with puromycin. At 750 mM KCl, 5 mM MgCl₂, 50 mM Tris·HCl, pH 7 5, up to 85% of all bound ribosomes are released from the membranes after incubation at room temperature with 1 mM puromycin. The ribosomes are released as subunits which are active in peptide synthesis if programmed with polyuridylic acid. The ribosome-denuded, or stripped, rough microsomes (RM) can be recovered as intact, essentially unaltered membranous vesicles Judging from the incorporation of [³H]puromycin into hot acid-insoluble material and from the release of [³H]leucine-labeled nascent polypeptide chains from bound ribosomes, puromycin coupling occurs almost as well at low (25–100 mM) as at high (500–1000 mM) KCl concentrations. Since puromycin-dependent ribosome release only occurs at high ionic strength, it appears that ribosomes are bound to membranes via two types of interactions: a direct one between the membrane and the large ribosomal subunit (labile at high KCl concentration) and an indirect one in which the nascent chain anchors the ribosome to the membrane (puromycin labile). The nascent chains of ribosomes specifically released by puromycin remain tightly associated with the stripped membranes. Some membrane-bound ribosomes (up to 40%) can be nondestructively released in high ionic strength media without puromycin; these appear to consist of a mixture of inactive ribosomes and ribosomes containing relatively short nascent chains. A fraction (~15%) of the bound ribosomes can only be released from membranes by exposure of RM to ionic conditions which cause extensive unfolding of ribosomal subunits, the nature and significance of these ribosomes is not clear.     

L25 functions as a conserved ribosomal docking site shared by nascent chain-associated complex and signal-recognition particle

The nascent chain-associated complex (NAC) is a dimeric protein complex of archaea and eukarya that interacts with ribosomes and translating polypeptide chains. We show that, in yeast, NAC and the signal-recognition particle (SRP) share the universally conserved ribosomal protein L25 as a docking site, which is in close proximity to the ribosomal exit tunnel. The amino-terminal segment of beta-NAC was found to be required for L25 binding. Purified NAC can prevent protein aggregation in vitro and thus shows certain properties of a molecular chaperone. Interestingly, the alpha-subunit of NAC interacts with the 54 kDa subunit of SRP. Consistent with a regulatory role of NAC in protein translocation into the endoplasmic reticulum (ER), we find that deletion of NAC results in an induction of the ER stress-response pathway. These results identify L25 as a conserved interaction platform for specific cytosolic factors that guide nascent polypeptides to their proper cellular destination.     

Biosynthesis of aldolase B by free ribosomes in rat liver

Free ribosomes and membrane-bound ribosomes were prepared from rat livers, and the contributions of these two types of ribosomes to the synthesis of aldolase B were studied by the immunoprecipitation of [3H]puromycin-labeled nascent peptides with a rabbit antibody to this enzyme. Although rat liver aldolase was recovered in both cytosolic and microsomal fractions by the fractionation of liver homogenate, the microsomal aldolase was immunologically identical with its cytosolic counterpart as confirmed by Ouchterlony immunodiffusion test. We examined the nascent peptide fractions prepared from free and bound ribosomes, and found that the nascent peptides of aldolase were mainly localized in free ribosomes. About 0.5% of the total nascent peptides of free ribosomes and 0.08% of those of bound ribosomes was aldolase. The site of synthesis of serum albumin was also examined as a reference standard by the immunoprecipitation of labeled nascent peptides, and the nascent peptides of this secretory protein were mainly associated with bound ribosomes, as reported by other workers. These observations confirm that aldolase B is mainly synthesized by free ribosomes in rat liver cells.     

Function of trigger factor and DnaK in multidomain protein folding: increase in yield at the expense of folding speed

Trigger factor and DnaK protect nascent protein chains from misfolding and aggregation in the E. coli cytosol, but how these chaperones affect the mechanism of de novo protein folding is not yet understood. Upon expression under chaperone-depleted conditions, multidomain proteins such as bacterial beta-galactosidase (beta-gal) and eukaryotic luciferase fold by a rapid but inefficient default pathway, tightly coupled to translation. Trigger factor and DnaK improve the folding yield of these proteins but markedly delay the folding process both in vivo and in vitro. This effect requires the dynamic recruitment of additional trigger factor molecules to translating ribosomes. While beta-galactosidase uses this chaperone mechanism effectively, luciferase folding in E. coli remains inefficient. The efficient cotranslational domain folding of luciferase observed in the eukaryotic system is not compatible with the bacterial chaperone system. These findings suggest important differences in the coupling of translation and folding between bacterial and eukaryotic cells.     

Co-translational modification of nascent immunoglobulin heavy and light chains

We have investigated the in vivo co-translational covalent modification of nascent immunoglobulin heavy and light chains. Nascent polypeptides were separated from completed polypeptides by ion-exchange chromatography of solubilized ribosomes on QAE-Sephadex. First, we have demonstrated that MPC 11 nascent heavy chains are quantitatively glycosylated very soon after the asparaginyl acceptor site passes through the membrane into the cisterna of the rough endoplasmic reticulum. Nonglycosylated completed heavy chains of various classes cannot be glycosylated after release from the ribosome, due either to rapid intramolecular folding and/or intermolecular assembly, which cause the acceptor site to become unavailable for the glycosylation enzyme. Second, we have shown that the formation of the correct intrachain disulfide loop within the first light chain domain occurs rapidly and quantitatively as soon as the appropriate cysteine residues of the nascent light chain pass through the membrane into the cisterna of the endoplasmic reticulum. The intrachain disulfide loop in the second or constant region domain of the light chain is not formed on nascent chains, because one of the cysteine residues involved in this disulfide bond does not pass through the endoplasmic reticulum membrane prior to chain completion and release from the ribosome. Third, we have demonstrated that some of the initial covalent assembly (formation of interchain disulfide bonds) occurs on nascent heavy chains prior to their release from the ribosome. The results are consistent with the pathway of covalent assembly of the cell line, in that completed light chains are assembled onto nascent heavy chains in MPC 11 cells (IgG₂b), where a heavy-light half molecule is the major initial covalent intermediate; and completed heavy chains are assembled onto nascent heavy chains in MOPC 21 cells (IgG₁), where a heavy chain dimer is the major initial disulfide linked intermediate.     

Co-Translational Membrane Targeting and Holo-Translocon Docking of Ribosomes Translating the SRP Receptor

Many integral membrane proteins are produced by translocon-associated ribosomes. The assembly of ribosomes translating membrane proteins on the translocons is mediated by a conserved system, composed of the signal recognition particle and its receptor (FtsY in Escherichia coli). FtsY is a peripheral membrane protein, and its role late during membrane protein targeting involves interactions with the translocon. However, earlier stages in the pathway have remained obscure, namely, how FtsY targets the membrane in vivo and where it initially docks. Our previous studies have demonstrated co-translational membrane-targeting of FtsY translation intermediates and identified a nascent FtsY targeting-peptide. Here, in a set of in vivo experiments, we utilized tightly stalled FtsY translation intermediates, pull-down assays and site-directed cross-linking, which revealed FtsY-nascent chain-associated proteins in the cytosol and on the membrane. Our results demonstrate interactions between the FtsY-translating ribosomes and cytosolic chaperones, which are followed by directly docking on the translocon. In support of this conclusion, we show that translocon over-expression increases dramatically the amount of membrane associated FtsY-translating ribosomes. The co-translational contacts of the FtsY nascent chains with the translocon differ from its post-translational contacts, suggesting a major structural maturation process. The identified interactions led us to propose a model for how FtsY may target the membrane co-translationally. On top of our past observations, the current results may add another tier to the hypothesis that FtsY acts stoichiometrically in targeting ribosomes to the membrane in a constitutive manner.