A complex from cultured Chinese hamster ovary cells containing nine aminoacyl‐tRNA synthetases

Mirande, Marc; Corre, Daniel Le; Waller, Jean‐Pierre

doi:10.1111/j.1432-1033.1985.tb08748.x

Cited by 94 publications

(52 citation statements)

References 28 publications

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“…Of the 13 activities assayed, aminoacyl-tRNA synthetases specific for arginine, aspartic acid, isoleucine, leucine, lysine, and proline co-eluted as a high molecular weight peak with an apparent molecular mass of ϳ10 6 Da (Fig. 1A), in complete agreement with the known composition of the stable core of the multienzyme complex from CHO cells (13). Arginyl-tRNA synthetase also had a second peak of activity in the low molecular weight region, as expected (27,28).…”

Section: Active Aminoacyl-trna Synthetases Are Present In Nuclei 31560supporting

confidence: 60%

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Active Aminoacyl-tRNA Synthetases Are Present in Nuclei as a High Molecular Weight Multienzyme Complex

Nathanson

Deutscher

2000

Journal of Biological Chemistry

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Recent studies suggest that aminoacylation of tRNA may play an important role in the transport of these molecules from the nucleus to the cytoplasm. However, there is almost no information regarding the status of active aminoacyl-tRNA synthetases within the nuclei of eukaryotic cells. Here we show that at least 13 active aminoacyl-tRNA synthetases are present in purified nuclei of both Chinese hamster ovary and rabbit kidney cells, although their steady-state levels represent only a small percentage of those found in the cytoplasm. Most interestingly, all the nuclear aminoacyl-tRNA synthetases examined can be isolated as part of a multienzyme complex that is more stable, and consequently larger, than the comparable complex isolated from the cytoplasm. These data directly demonstrate the presence of active aminoacyl-tRNA synthetases in mammalian cell nuclei. Moreover, their unexpected structural organization raises important questions about the functional significance of these multienzyme complexes and whether they might play a more direct role in nuclear to cytoplasmic transport of tRNAs.

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Section: Active Aminoacyl-trna Synthetases Are Present In Nuclei 31560supporting

confidence: 60%

“…Aminoacyl-tRNA synthetases, as well as elongation factor 1 (EF1), 1 also could be detected in nuclei by immunochemical methods (13)(14)(15)(16). However, these studies provided no information as to whether the nuclear-localized synthetases were active.…”

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Active Aminoacyl-tRNA Synthetases Are Present in Nuclei as a High Molecular Weight Multienzyme Complex

Nathanson

Deutscher

2000

Journal of Biological Chemistry

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“…The AspRS extension carries a stretch of 10 alkaline amino acids over amino acids 31 -50, which, as was demonstrated in the case of mammalian LysRS [35], may account for the strong retention of the enzyme on heparin, a property common to eukaryotic aaRS [34]. This capacity for polyanionbinding may promote, as proposed, interactions with nucleic acids or negatively charged structures inside the cell, favouring enzyme compartmentalisation [39].…”

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confidence: 95%

“…N-terminal extensions appear to be a feature characteristic of eucaryotic aaRS, whereas they could not be detected in prokaryotes; their function in yeast enzymes remains hypothetic since, as shown at least for GlmRS and MetRS, these sequences are not required for cell viability [36, 381. They do not appear to be involved in supramolecular formation as could be shown for mammalian aaRS [39], since the existence of such multienzyme complexes has not yet been demonstrated for yeast. The AspRS extension carries a stretch of 10 alkaline amino acids over amino acids 31 -50, which, as was demonstrated in the case of mammalian LysRS [35], may account for the strong retention of the enzyme on heparin, a property common to eukaryotic aaRS [34].…”

Section: Sdsjpage Analysis Of Crude Extracts For Their Content Of Cmentioning

confidence: 99%

Cytoplasmic aspartyl‐tRNA synthetase from Saccharomyces cerevisiae

Eriani

Prévost

Kern

et al. 1991

European Journal of Biochemistry

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Aspartyl-tRNA synthetase (AspRS) from yeast, a homodimer of 125 kDa, was shortened by several residues from the C-and N-termini, via site-directed mutagenesis, to examine the contribution of the removed peptides to the enzyme properties. This study showed that the N-terminal sequence up to amino acid 70 (which confers peculiar ionic properties to the protein) is dispensable for activity. Domains located beyond amino acid 70 appeared to have increasing catalytic importance; the removal of 80 or 90 residues affected the K, values for ATP and deletions of 101 or 140 amino acids profoundly modified the physicochemical properties of AspRS, and by consequence, its structural organisation (extraction of the mutated proteins out of the cells required the presence of SDS). On the C-terminal side, very limited modifications readily affected the enzyme properties. Deletion of as few as three residues increased the K , for ATP and reduced the aminoacylation k,,, as well as the thermostability of the adenylate synthesis activity; the k,,, of this step was impaired after deletion of two further residues. Finally, shortening the C-terminal decapeptide completely inactivated AspRS, whilst affecting neither its affinity for tRNAAsp nor its dimerisation capacity. These data reveal the role of the C-terminal decapeptide as a determinant in both reactions catalysed by AspRS. This peptide is involved in ATP binding, stabilising the functional conformation of the amino-acid-activating domain and probably maintaining the tRNA-acceptor end in a reactive position with regard to the activated amino acid.Translation of the classical genetic code into polypeptides of defined sequences depends on the correct aminoacylation of tRNA and, by consequence, on the high specificity for substrates and catalysis of the aminoacyl-tRNA synthetases (aaRS). Important investigations of these enzymes have been undertaken by site-directed mutagenesis, combined with crystallographic analysis of the proteins or their complexes with specific tRNA [l -91, to establish the structural elements responsible for their activities. These enzymes, in spite of catalysing the same type of reactions, diverge greatly in their subunit size and oligomeric structure, and show very limited sequence similarity. However, half of the known aaRS contain the two short common sequences KMSKS and HIGH, which are now generally considered, as demonstrated for some of these enzymes, to be close to the tRNA-acceptor end, and to have important interactions with ATP 14, 101. The crystallographic structure of TyrRS, MetRS and GlnRS from Escherichiu coli [5, 6, 111 show that these consensus motifs are present in the so-called Rossmann fold, a domain that binds ATP. The aim of the study reported here for yeast AspRS was

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“…In eukaryotes, MSCs tend to be larger than those discovered in bacteria and archaea and also perform a wider range of functions that include both aminoacylation and noncanonical roles beyond translation (6 -9). The mammalian MSC, purified from various mammalian tissues (10 -13) and from cultured Chinese hamster ovary (CHO) (14) and murine erythroleukemia cells (15), is by far the largest known MSC (16 -19). This complex is composed of eleven polypeptides including the bi-functional glutamyl-prolyl-tRNA synthetase (GluProRS), seven monospecific aspartyl-, arginyl-, glutaminyl-, lysyl-, methionyl-, leucyl-, and isoleucyl-tRNA synthetases (AspRS, ArgRS, GlnRS, LysRS, MetRS, LeuRS, and IleRS, respectively); and three nonsynthetase protein factors, p18, p38, and p43 (20,21,22).…”

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An Archaeal tRNA-Synthetase Complex that Enhances Aminoacylation under Extreme Conditions

Godinić-Mikulčić

Jarić

Hausmann

et al. 2011

Journal of Biological Chemistry

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Aminoacyl-tRNA synthetases (aaRSs) play an integral role in protein synthesis, functioning to attach the correct amino acid with its cognate tRNA molecule. AaRSs are known to associate into higher-order multi-aminoacyl-tRNA synthetase complexes (MSC) involved in archaeal and eukaryotic translation, although the precise biological role remains largely unknown. To gain further insights into archaeal MSCs, possible proteinprotein interactions with the atypical Methanothermobacter thermautotrophicus seryl-tRNA synthetase (MtSerRS) were investigated. Yeast two-hybrid analysis revealed arginyl-tRNA synthetase (MtArgRS) as an interacting partner of MtSerRS. Surface plasmon resonance confirmed stable complex formation, with a dissociation constant (K D ) of 250 nM. Formation of the MtSerRS⅐MtArgRS complex was further supported by the ability of GST-MtArgRS to co-purify MtSerRS and by coelution of the two enzymes during gel filtration chromatography. The MtSerRS⅐MtArgRS complex also contained tRNA Arg , consistent with the existence of a stable ribonucleoprotein complex active in aminoacylation. Steady-state kinetic analyses revealed that addition of MtArgRS to MtSerRS led to an almost 4-fold increase in the catalytic efficiency of serine attachment to tRNA, but had no effect on the activity of MtArgRS. Further, the most pronounced improvements in the aminoacylation activity of MtSerRS induced by MtArgRS were observed under conditions of elevated temperature and osmolarity. These data indicate that formation of a complex between MtSerRS and MtArgRS provides a means by which methanogenic archaea can optimize an early step in translation under a wide range of extreme environmental conditions. Aminoacyl-tRNA synthetases (aaRSs)2 catalyze the specific coupling of amino acids with their cognate tRNAs to produce aminoacyl-tRNAs (aa-tRNAs), which serve as starting materials for the biosynthesis of proteins. Aa-tRNA synthesis occurs in two steps: amino acid activation at the expense of ATP followed by the aminoacylation of tRNA (1). Although for most aaRSs the formation of aminoacyl-AMP does not require tRNA, cognate tRNA is necessary for amino acid activation by ArgRS, GlnRS, GluRS, and LysRS1 enzymes from many organisms (2). Based on structural features of their active sites, aaRSs can be divided into two classes, which comprise 10 members each (3). In addition, an unusual form of LysRS is found in class I (2), while class II also includes the noncanonical synthetases PylRS and SepRS (4).In all three domains of life, subsets of aaRSs have been shown to associate into higher-order multi-aminoacyl-tRNA synthetase complexes (MSCs). These complexes are distinctive compared with other macromolecular protein complexes, because their components are enzymes that carry out similar catalytic reactions simultaneously, and only some aaRSs are involved (5). In eukaryotes, MSCs tend to be larger than those discovered in bacteria and archaea and also perform a wider range of functions that include both aminoacylation and noncanonical roles b...

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A complex from cultured Chinese hamster ovary cells containing nine aminoacyl‐tRNA synthetases

Cited by 94 publications

References 28 publications

Active Aminoacyl-tRNA Synthetases Are Present in Nuclei as a High Molecular Weight Multienzyme Complex

Active Aminoacyl-tRNA Synthetases Are Present in Nuclei as a High Molecular Weight Multienzyme Complex

Cytoplasmic aspartyl‐tRNA synthetase from Saccharomyces cerevisiae

An Archaeal tRNA-Synthetase Complex that Enhances Aminoacylation under Extreme Conditions

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