Co-translational Process Research Articles

Probing ribosome-nascent chain complexes produced in vivo by NMR spectroscopy

The means by which a polypeptide chain acquires its unique 3-D structure is a fundamental question in biology. During its synthesis on the ribosome, a nascent chain (NC) emerges vectorially and will begin to fold in a cotranslational fashion. The complex environment of the cell, coupled with the gradual emergence of the ribosome-tethered NC during its synthesis, imposes conformational restraints on its folding landscape that differ from those placed on an isolated protein when stimulated to fold following denaturation in solution. To begin to examine cotranslational folding as it would occur within a cell, we produce highly selective, isotopically labeled NCs bound to isotopically silent ribosomes in vivo. We then apply NMR spectroscopy to study, at a residue specific level, the conformation of NCs consisting of different fractional lengths of the polypeptide chain corresponding to a given protein. This combined approach provides a powerful means of generating a series of snapshots of the folding of the NC as it emerges from the ribosome. Application of this strategy to the NMR analysis of the progressive synthesis of an Ig-like domain reveals the existence of a partially folded ribosome-bound species that is likely to represent an intermediate species populated during the cotranslational folding process.

Cotranslational Proteolysis Dominates Glutathione Homeostasis to Support Proper Growth and Development

The earliest proteolytic event affecting most proteins is the excision of the initiating Met (NME). This is an essential and ubiquitous cotranslational process tightly regulated in all eukaryotes. Currently, the effects of NME on unknown complex cellular networks and the ways in which its inhibition leads to developmental defects and cell growth arrest remain poorly understood. Here, we provide insight into the earliest molecular mechanisms associated with the inhibition of the NME process in Arabidopsis thaliana. We demonstrate that the developmental defects induced by NME inhibition are caused by an increase in cellular proteolytic activity, primarily induced by an increase in the number of proteins targeted for rapid degradation. This deregulation drives, through the increase of the free amino acids pool, a perturbation of the glutathione homeostasis, which corresponds to the earliest limiting, reversible step promoting the phenotype. We demonstrate that these effects are universally conserved and that the reestablishment of the appropriate glutathione status restores growth and proper development in various organisms. Finally, we describe a novel integrated model in which NME, protein N-alpha-acylation, proteolysis, and glutathione homeostasis operate in a sequentially regulated mechanism that directs both growth and development.

Statics of the Ribosomal Exit Tunnel: Implications for Cotranslational Peptide Folding, Elongation Regulation, and Antibiotics Binding

A sophisticated interplay between the static properties of the ribosomal exit tunnel and its functional role in cotranslational processes is revealed by constraint counting on topological network representations of large ribosomal subunits from four different organisms. As for the global flexibility characteristics of the subunit, the results demonstrate a conserved stable structural environment of the tunnel. The findings render unlikely that deformations of the tunnel move peptides down the tunnel in an active manner. Furthermore, the stable environment rules out that the tunnel can adapt widely so as to allow tertiary folding of nascent chains. Nevertheless, there are local zones of flexible nucleotides within the tunnel, between the peptidyl transferase center and the tunnel constriction, and at the tunnel exit. These flexible zones strikingly agree with previously identified folding zones. As for cotranslational elongation regulation, flexible residues in the β-hairpin of the ribosomal L22 protein were verified, as suggested previously based on structural results. These results support the hypothesis that L22 can undergo conformational changes that regulate the tunnel voyage of nascent polypeptides. Furthermore, rRNA elements, for which conformational changes have been observed upon interaction of the tunnel wall with a nascent SecM peptide, are less strongly coupled to the subunit core. Sequences of coupled rigid clusters are identified between the tunnel and some of these elements, suggesting signal transmission by a domino-like mechanical coupling. Finally, differences in the flexibility of the glycosidic bonds of bases that form antibiotics-binding crevices within the peptidyl transferase center and the tunnel region are revealed for ribosomal structures from different kingdoms. In order to explain antibiotics selectivity, action, and resistance, according to these results, differences in the degrees of freedom of the binding regions may need to be considered.

The Cotranslational Maturation Program for the Type II Membrane Glycoprotein Influenza Neuraminidase

The earliest steps in nascent protein maturation greatly affect its overall efficiency. Constraints placed on maturing proteins at these early stages limit available conformations and help to direct the native maturation process. For type II membrane proteins, these cotranslational constraints include N- and C-terminal membrane tethering, chaperone binding, and disulfide bond formation. The cotranslational maturation process for the type II membrane glycoprotein influenza neuraminidase (NA) was investigated to provide a deeper understanding of these initial endoplasmic reticulum events. The type II orientation provides experimental advantages to monitor the first maturation steps. Calnexin was shown to cotranslationally interact with NA prior to calreticulin. These interactions were required for the efficient maturation of NA as it prematurely formed intramolecular disulfides and aggregated when calnexin and calreticulin interactions were abolished. Lectin chaperone binding slowed the NA maturation process, increasing its fidelity. Carbohydrates were required for NA maturation in a regio-specific manner. A subset of NA formed intermolecular disulfides and oligomerized cotranslationally. This fraction increased in the absence of calnexin and calreticulin binding. NA dimerization also occurred for an NA mutant lacking the critical large loop disulfide bond, indicating that dimerization did not require proper NA oxidation. The strict evaluation of proper maturation carried out by the quality control machinery was instilled at the tetramerization step. This study illustrates the type II membrane protein maturation process and shows how important cotranslational events contribute to the proper cellular maturation of glycoproteins.

Yeast Mitochondrial Biogenesis: A Role for the PUF RNA-Binding Protein Puf3p in mRNA Localization

The asymmetric localization of mRNA plays an important role in coordinating posttranscriptional events in eukaryotic cells. We investigated the peripheral mitochondrial localization of nuclear-encoded mRNAs (MLR) in various conditions in which the mRNA binding protein context and the translation efficiency were altered. We identified Puf3p, a Pumilio family RNA-binding protein, as the first trans-acting factor controlling the MLR phenomenon. This allowed the characterization of two classes of genes whose mRNAs are translated to the vicinity of mitochondria. Class I mRNAs (256 genes) have a Puf3p binding motif in their 3'UTR region and many of them have their MLR properties deeply affected by PUF3 deletion. Conversely, mutations in the Puf3p binding motif alter the mitochondrial localization of BCS1 mRNA. Class II mRNAs (224 genes) have no Puf3p binding site and their asymmetric localization is not affected by the absence of PUF3. In agreement with a co-translational import process, we observed that the presence of puromycin loosens the interactions between most of the MLR-mRNAs and mitochondria. Unexpectedly, cycloheximide, supposed to solidify translational complexes, turned out to destabilize a class of mRNA-mitochondria interactions. Classes I and II mRNAs, which are therefore transported to the mitochondria through different pathways, correlated with different functional modules. Indeed, Class I genes code principally for the assembly factors of respiratory chain complexes and the mitochondrial translation machinery (ribosomes and translation regulators). Class II genes encode proteins of the respiratory chain or proteins involved in metabolic pathways. Thus, MLR, which is intimately linked to translation control, and the activity of mRNA-binding proteins like Puf3p, may provide the conditions for a fine spatiotemporal control of mitochondrial protein import and mitochondrial protein complex assembly. This work therefore provides new openings for the global study of mitochondria biogenesis.

Subcellular Localization and Physiological Significance of Intracellular Mannan-binding Protein

Mannan-binding protein (MBP) is a C-type mammalian lectin specific for mannose and N-acetylglucosamine. MBP is mainly synthesized in the liver and occurs naturally in two forms, serum MBP (S-MBP) and intracellular MBP (I-MBP). S-MBP activates complement in association with MBP-associated serine proteases via the lectin pathway. Despite our previous study (Mori, K., Kawasaki, T., and Yamashina, I. (1984) Arch. Biochem. Biophys. 232, 223-233), the subcellular localization of I-MBP and its functional implication have not been clarified yet. Here, as an extension of our previous studies, we have demonstrated that the expression of human MBP cDNA reproduces native MBP differentiation of S-MBP and I-MBP in human hepatoma cells. I-MBP shows distinct accumulation in cytoplasmic granules, and is predominantly localized in the endoplasmic reticulum (ER) and involved in COPII vesicle-mediated ER-to-Golgi transport. However, the subcellular localization of either a mutant (C236S/C244S) I-MBP, which lacks carbohydrate-binding activity, or the wild-type I-MBP in tunicamycin-treated cells shows an equally diffuse cytoplasmic distribution, suggesting that the unique accumulation of I-MBP in the ER and COPII vesicles is mediated by an N-glycan-lectin interaction. Furthermore, the binding of I-MBP with glycoprotein intermediates occurs in the ER, which is carbohydrate- and pH-dependent, and is affected by glucose-trimmed high-mannose-type oligosaccharides. These results strongly indicate that I-MBP may function as a cargo transport lectin facilitating ER-to-Golgi traffic in glycoprotein quality control.

Posttranslational N-glycosylation of the hepatitis B virus large envelope protein

BackgroundThe addition of N-linked glycans to proteins is normally a cotranslational process that occurs during translocation of the nascent protein to the endoplasmic reticulum. Here, we report on an exception to this rule occurring on the hepatitis B virus (HBV) large L envelope protein that is a subject to co-plus posttranslational N-glycosylation.ResultsBy using an improved detection system, we identified so far unrecognized, novel isoforms of L. Based on mutational analyses, the use of N-glycosylation inhibitors, and pulse-chase studies, we showed that these isoforms are due to posttranslational N-glycan addition to the asparagines 4 and 112 within the preS domain of L. While an inhibition of N-glycosylation and glycan trimming profoundly blocked virus assembly and release, the posttranslational N-glycosylation of L itself was found to be dispensable for HBV morphogenesis.ConclusionThese data together with previous results implicate that the N-glycosylation requirements of virion release are due to functional inhibition of cell glycoproteins engaged by HBV.

Biogenesis and Topology of the Secretory Na+−K+−2Cl- Cotransporter (NKCC1) Studied in Intact Mammalian Cells

The "secretory" Na+-K+-2Cl- cotransporter (NKCC1) is a member of a small gene family with nine homologues in vertebrates. Of these, seven are known to be electroneutral chloride transporters. These transporters play a number of important physiological roles related to salt and water homeostasis and the control of intracellular chloride levels. Hydropathy analyses suggest that all of these transporters have a similar transmembrane topology consisting of relatively large intracellular N and C termini and a central hydrophobic domain containing 12 membrane-spanning segments (MSSs). In recent experiments from our laboratory [Gerelsaikhan, T., and Turner, R. J. (2000) J. Biol. Chem. 275, 40471-40477], we employed an in vitro translation system to confirm that each of the putative MSSs of NKCC1 was capable of membrane integration in a manner consistent with a 12 MSS model. Here, we extend that work to the study of the biogenesis of NKCC1 in intact cells. We employ a truncation mutant approach that allows us to monitor this process quantitatively as successive MSSs are synthesized. While the results presented here confirm the 12 MSS model, they also indicate that the integration of NKCC1 into the membrane does not occur via a simple cotranslational process. In particular, we demonstrate that two MSSs, the second and sixth, require the presence of downstream sequence to efficiently integrate into the membrane.

Characterization of N‐glycosylation consensus sequences in the Kv3.1 channel

N-Glycosylation is a cotranslational and post-translational process of proteins that may influence protein folding, maturation, stability, trafficking, and consequently cell surface expression of functional channels. Here we have characterized two consensus N-glycosylation sequences of a voltage-gated K+ channel (Kv3.1). Glycosylation of Kv3.1 protein from rat brain and infected Sf9 cells was demonstrated by an electrophoretic mobility shift assay. Digestion of total brain membranes with peptide N glycosidase F (PNGase F) produced a much faster-migrating Kv3.1 immunoband than that of undigested brain membranes. To demonstrate N-glycosylation of wild-type Kv3.1 in Sf9 cells, cells were treated with tunicamycin. Also, partially purified proteins were digested with either PNGase F or endoglycosidase H. Attachment of simple-type oligosaccharides at positions 220 and 229 was directly shown by single (N229Q and N220Q) and double (N220Q/N229Q) Kv3.1 mutants. Functional measurements and membrane fractionation of infected Sf9 cells showed that unglycosylated Kv3.1s were transported to the plasma membrane. Unitary conductance of N220Q/N229Q was similar to that of the wild-type Kv3.1. However, whole cell currents of N220Q/N229Q channels had slower activation rates, and a slight positive shift in voltage dependence compared to wild-type Kv3.1. The voltage dependence of channel activation for N229Q and N220Q was much like that for N220Q/N229Q. These results demonstrate that the S1-S2 linker is topologically extracellular, and that N-glycosylation influences the opening of the voltage-dependent gate of Kv3.1. We suggest that occupancy of the sites is critical for folding and maturation of the functional Kv3.1 at the cell surface.

Posttranslational myristoylation of caspase-activated p21-activated protein kinase 2 (PAK2) potentiates late apoptotic events

p21-activated protein kinase (PAK) 2 is a small GTPase-activated serine/threonine kinase regulating various cytoskeletal functions and is cleaved by caspase-3 during apoptosis. We demonstrate that the caspase-cleaved PAK2 C-terminal kinase fragment (C-t-PAK2) is posttranslationally myristoylated, although myristoylation is typically a cotranslational process. Myristoylation and an adjacent polybasic domain of C-t-PAK2 are sufficient to redirect EGFP from the cytosol to membrane ruffles and internal membranes. Membrane localization and the ability of C-t-PAK2 to induce cell death are significantly reduced when myristoylation is abolished. In addition, the proper myristoylation-dependent membrane localization of C-t-PAK2 significantly increased signaling through the stress-activated c-Jun N-terminal kinase signaling pathway, which often regulates apoptosis. Interestingly, C-t-PAK2 promoted cell death without compromising mitochondrial integrity. Posttranslational myristoylation of caspase-cleaved proteins involved in cytoskeletal dynamics (e.g., PAK2, actin, and gelsolin) might be part of a unique series of mechanisms involved in the regulation of the later events of apoptosis.

Genomic and functional analyses reveal a distinct chaperone network linked to protein biosynthesis in eukaryotic cells

Molecular chaperones assist the folding of newly translated and stress-denatured proteins. In prokaryotes, overlapping sets of chaperones mediate both processes. In contrast, we find here that eukaryotes have evolved distinct chaperone networks to carry out these functions. A systems approach combining genomic and functional analyses indicate that in addition to stress-inducible chaperones, which protect cells from environmental stress, eukaryotes contain a stress-repressed chaperone network that is dedicated to protein biogenesis. These stress-repressed chaperones are transcriptionally, functionally and physically linked to the translational apparatus and associate with nascent polypeptides emerging from the ribosome. Consistent with a function in de novo protein folding, impairment of the translation-linked chaperone network renders cells sensitive to misfolding in the context of protein synthesis but not in the context of environmental stress. The emergence of a translation-linked chaperone network likely underlies the elaborate co-translational folding process necessary for the evolution of larger multidomain proteins characteristic of eukaryotic cells. Supported by NIH GM56433

Two Hrd1p homologues in the yeast Yarrowia lipolytica which act in different pathways

The endoplasmic reticulum associated degradation (ERAD) is a process widespread in eukaryotes that enable cells to get rid of unfolded or unassembled polypeptides which jam the endoplasmic reticulum compartment. In order to improve understanding of the initial steps of the secretory pathway and their relationship, we focused on components of the ERAD ubiquitylation machinery in the yeast Yarrowia lipolytica. Two Hrd1p homologues, Hrd1p and Hrh1p, were identified in Y. lipolytica. A study of the fate of the heterologous CPY* reporter protein showed that YlHrd1p is involved in the elimination of this misfolded polypeptide, while YlHrh1p is not. Moreover, the different phenotypic pattern displayed by Deltahrd1 and Deltahrh1 cells suggests that the two putative E3 enzymes function in separate ways. Our results bring some evidence of a coupling between the ERAD pathway and the co-translational translocation process and show that studies in Y. lipolytica can give new insights into events that take place in the ER.

Distinct yet linked: chaperone networks in the eukaryotic cytosol

Although one subset of chaperones is induced by heat stress, a distinct group fails to respond in the same manner. Recent work reveals that this latter group is linked to the translational apparatus and functions in co-translational processes.

Systems Analyses Reveal Two Chaperone Networks with Distinct Functions in Eukaryotic Cells

Molecular chaperones assist the folding of newly translated and stress-denatured proteins. In prokaryotes, overlapping sets of chaperones mediate both processes. In contrast, we find that eukaryotes evolved distinct chaperone networks to carry out these functions. Genomic and functional analyses indicate that in addition to stress-inducible chaperones that protect the cellular proteome from stress, eukaryotes contain a stress-repressed chaperone network that is dedicated to protein biogenesis. These stress-repressed chaperones are transcriptionally, functionally, and physically linked to the translational apparatus and associate with nascent polypeptides emerging from the ribosome. Consistent with a function in de novo protein folding, impairment of the translation-linked chaperone network renders cells sensitive to misfolding in the context of protein synthesis but not in the context of environmental stress. The emergence of a translation-linked chaperone network likely underlies the elaborate cotranslational folding process necessary for the evolution of larger multidomain proteins characteristic of eukaryotic cells.

Precursors of Novel Gla-Containing Conotoxins Contain a Carboxy-Terminal Recognition Site That Directs γ-Carboxylation,

Vitamin K-dependent gamma-glutamyl carboxylase catalyzes the conversion of glutamyl residues to gamma-carboxyglutamate. Its substrates include vertebrate proteins involved in blood coagulation, bone mineralization, and signal transduction and invertebrate ion channel blockers known as conotoxins. Substrate recognition involves a recognition element, the gamma-carboxylation recognition site, typically located within a cleavable propeptide preceding the targeted glutamyl residues. We have purified two novel gamma-carboxyglutamate-containing conotoxins, Gla-TxX and Gla-TxXI, from the venom of Conus textile. Their cDNA-deduced precursors have a signal peptide but no apparent propeptide. Instead, they contain a C-terminal extension that directs gamma-carboxylation but is not found on the mature conotoxin. A synthetic 13-residue "postpeptide" from the Gla-TxXI precursor reduced the K(m) for the reaction of the Conus gamma-carboxylase with peptide substrates, including FLEEL and conantokin-G, by up to 440-fold, regardless of whether it was positioned at the N- or C-terminal end of the mature toxin. Comparison of the postpeptides to propeptides from other conotoxins suggested some common elements, and amino acid substitutions of these residues perturbed gamma-carboxylation of the Gla-TxXI peptide. The demonstration of a functional and transferable C-terminal postpeptide in these conotoxins indicates the presence of the gamma-carboxylation recognition site within the postpeptide and defines a novel precursor structure for vitamin K-dependent polypeptides. It also provides the first formal evidence to prove that gamma-carboxylation occurs as a post-translational rather than a cotranslational process.

The Cotranslational Maturation of the Type I Membrane Glycoprotein Tyrosinase: The Heat Shock Protein 70 System Hands Off to the Lectin-based Chaperone System

The maturation of eukaryotic secretory cargo initiates cotranslationally and cotranslocationally as the polypeptide chain emerges into the endoplasmic reticulum lumen. Here, we characterized the cotranslational maturation pathway for the human type I membrane glycoprotein tyrosinase. To recapitulate the cotranslational events, including glycosylation, signal sequence cleavage, chaperone binding, and oxidation, abbreviated transcripts lacking a stop codon were in vitro translated in the presence of semipermeabilized melanocyte membranes. This created a series of ribosome/translocon-arrested chains of increasing lengths, simulating intermediates in the cotranslational folding process. Initially, nascent chains were found to associate with the heat shock protein (Hsp) 70 family member BiP. As the nascent chains elongated and additional glycans were transferred, BiP binding rapidly decreased and the lectin-based chaperone system was recruited in its place. The lectin chaperone calnexin bound to the nascent chain after the addition of two glycans, and calreticulin association followed upon the addition of a third. The glycan-specific oxidoreductase ERp57 was cross-linked to tyrosinase when calnexin and calreticulin were associated. This timing coincided with the formation of disulfide bonds within tyrosinase and the cleavage of its signal sequence. Therefore, tyrosinase maturation initiates cotranslationally with the Hsp70 system and is handed off to the lectin chaperone system that first uses calnexin before calreticulin. Interestingly, divergence in the maturation pathways of wild-type and mutant albino tyrosinase can already be observed for translocon-arrested nascent chains.

Posttranslational N-glycosylation takes place during the normal processing of human coagulation factor VII

N-glycosylation is normally a cotranslational process that occurs during translocation of the nascent protein to the endoplasmic reticulum. In the present study, however, we demonstrate posttranslational N-glycosylation of recombinant human coagulation factor VII (FVII) in CHO-K1 and 293A cells. Human FVII has two N-glycosylation sites (N145 and N322). Pulse-chase labeled intracellular FVII migrated as two bands corresponding to FVII with one and two N-glycans, respectively. N-glycosidase treatment converted both of these band into a single band, which comigrated with mutated FVII without N-glycans. Immediately after pulse, most labeled intracellular FVII had one N-glycan, but during a 1-h chase, the vast majority was processed into FVII with two N-glycans, demonstrating posttranslational N-glycosylation of FVII. Pulse-chase analysis of N-glycosylation site knockout mutants demonstrated cotranslational glycosylation of N145 but primarily or exclusively posttranslational glycosylation of N322. The posttranslational N-glycosylation appeared to take place in the same time frame as the folding of nascent FVII into a secretion-competent conformation, indicating a link between the two processes. We propose that the cotranslational conformation(s) of FVII are unfavorable for glycosylation at N332, whereas a more favorable conformation is obtained during the posttranslational folding. This is the first documentation of posttranslational N-glycosylation of a non-modified protein in mammalian cells with an intact N-glycosylation machinery. Thus, the present study demonstrates that posttranslational N-glycosylation can be a part of the normal processing of glycoproteins.

Expression of methionine aminopeptidase 2, N-myristoyltransferase, and N-myristoyltransferase inhibitor protein 71 in HT29

Protein myristoylation is a co-translational process, catalyzed by N-myristoyltransferase (NMT) that occurs after the initiating methionine is removed by methionine aminopeptidase (MetAP). The enzymes NMT and MetAP play a major role in the process of myristoylation of oncoproteins including the c-src family. In this study, we examined the levels of expression of MetAP2, NMT, and NMT inhibitor protein 71 (NIP71) in human colon cancer cell lines (HCCLs). We examined the influence of cell density on the expression of the above proteins in HT29 cells. Western blot analysis of MetAP2 and NMT demonstrated higher levels of protein expression in low density of HT29 while low expression in high density was observed. In addition, we observed that NIP71 and pp60 c-src expressions were dependent on the cell density of HT29. This is the first study demonstrating the expression of MetAP2, NMT, pp60 c-src, and NIP71 in HCCLs.

Heterologous expression and biophysical characterization of soluble oligosaccharyl transferase subunits

Oligosaccharyl transferase (OT) catalyzes the first committed step in N-linked protein glycosylation, a co-translational process that occurs in the lumen of the endoplasmic reticulum. The yeast Saccharomyces cerevisiae enzyme complex comprises nine integral membrane proteins, five of which are essential for catalysis. Due to the challenges with purifying the active enzyme complex for detailed biophysical studies, a systematic study to express, isolate, and characterize the soluble domains of three of the largest subunits in the complex (Nlt1p, Wbp1p, and Swp1p) is reported. The proteins are expressed using the lytic baculovirus expression system and the new constructs are well behaved, monomeric in solution, and glycosylated. Two of the proteins interact with each other as seen by gel filtration and circular dichroism. This study provides a framework to study the roles of these three essential subunits of the eukaryotic OT complex.

Ribosome binding to the Oxa1 complex facilitates co-translational protein insertion in mitochondria.

The Oxa1 translocase of the mitochondrial inner membrane facilitates the insertion of both mitochondrially and nuclear-encoded proteins from the matrix into the inner membrane. Most mitochondrially encoded proteins are hydrophobic membrane proteins which are integrated into the lipid bilayer during their synthesis on mitochondrial ribosomes. The molecular mechanism of this co-translational insertion process is unknown. Here we show that the matrix-exposed C-terminus of Oxa1 forms an alpha-helical domain that has the ability to bind to mitochondrial ribosomes. Deletion of this Oxa1 domain strongly diminished the efficiency of membrane insertion of subunit 2 of cytochrome oxidase, a mitochondrially encoded substrate of the Oxa1 translocase. This suggests that co-translational membrane insertion of mitochondrial translation products is facilitated by a physical interaction of translation complexes with the membrane-bound translocase.