The N-terminal Domain of Mammalian Lysyl-tRNA Synthetase Is a Functional tRNA-binding Domain

Francin, Mathilde; Kamińska, M.; Kerjan, Pierre; Mirande, Marc

doi:10.1074/jbc.m109759200

Cited by 103 publications

(142 citation statements)

References 51 publications

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“…Similarly, no complexes were detected between tRK1 and Eno1p (data not shown). tRK1 interacted with preMsk1p with an apparent dissociation constant Kd = 180 ± 20 nM (dimer concentration), comparable with Kd reported for complexes of lysyl-tRNA synthetases (LysRS) with noncognate tRNAs (Francin et al 2002). The affinity of tRK1 to Eno2p (apparent Kd = 2.5 ± 0.2 µM) was one order lower than preMsk1p.…”

Section: Eno2p Binds To Trk1 and Enhances Formation Of Trk1-premsk1p mentioning

confidence: 56%

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A glycolytic enzyme, enolase, is recruited as a cofactor of tRNA targeting toward mitochondria in Saccharomyces cerevisiae

Entelis¹,

Brandina²,

Kamenski³

et al. 2006

Genes Dev.

158

134

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In many organisms, mitochondria import nuclear DNA-encoded small RNAs. In yeast Saccharomyces cerevisiae, one out of two cytoplasmic isoacceptor tRNAs Lys is partially addressed into the organelle. Mitochondrial targeting of this tRNA was shown to depend on interaction with the precursor of mitochondrial lysyl-tRNA synthetase, preMsk1p. However, preMsk1p alone was unable to direct tRNA targeting, suggesting the existence of additional protein factor(s). Here, we identify the glycolytic enzyme, enolase, as such a factor. We demonstrate that recombinant enolase and preMSK1p are sufficient to direct tRNA import in vitro and that depletion of enolase inhibits tRNA import in vivo. Enzymatic and tRNA targeting functions of enolase appear to be independent. Three newly characterized properties of the enolase can be related to its novel function: (1) specific affinity to the imported tRNA, (2) the ability to facilitate formation of the complex between preMsk1p and the imported tRNA, and (3) partial targeting toward the mitochondrial outer membrane. We propose a model suggesting that the cell exploits mitochondrial targeting of the enolase in order to address the tRNA toward peri-mitochondrially synthesized preMsk1p. Our results indicate an alternative molecular chaperone function of glycolytic enzyme enolase in tRNA mitochondrial targeting.

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Section: Eno2p Binds To Trk1 and Enhances Formation Of Trk1-premsk1p mentioning

confidence: 56%

“…Separation was done in native 6% PAGE, 20 mM Tris-borate buffer (pH 8.3), and 5% Glycerol as described (Kaminska et al 2000). Free and bound tRNA was quantified by Fuji PhosphorImager, and apparent dissociation constants for tRNA-protein complexes were estimated as in Francin et al (2002).…”

Section: Isolation and Analysis Of Rnamentioning

confidence: 99%

A glycolytic enzyme, enolase, is recruited as a cofactor of tRNA targeting toward mitochondria in Saccharomyces cerevisiae

Entelis¹,

Brandina²,

Kamenski³

et al. 2006

Genes Dev.

158

134

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“…43). For example, Arc1p/p43 acts in trans to enhance catalytic activity (44,45), whereas extensions of eukaryotic aaRSs can enhance both specificity (46) and aminoacylation in cis (38). The interaction between LeuRS and ProRS described here falls into neither of these categories, with an aaRS rather than an accessory factor providing catalytic enhancement in trans.…”

Section: Discussionmentioning

confidence: 82%

Association between Archaeal Prolyl- and Leucyl-tRNA Synthetases Enhances tRNAPro Aminoacylation

Prætorius-Ibba¹,

Rogers²,

Samson³

et al. 2005

Journal of Biological Chemistry

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Aminoacyl-tRNA synthetase-containing complexes have been identified in different eukaryotes, and their existence has also been suggested in some Archaea. To investigate interactions involving aminoacyl-tRNA synthetases in Archaea, we undertook a yeast two-hybrid screen for interactions between Methanothermobacter thermautotrophicus proteins using prolyl-tRNA synthetase (ProRS) as the bait. Interacting proteins identified included components of methanogenesis, protein-modifying factors, and leucyl-tRNA synthetase (LeuRS). The association of ProRS with LeuRS was confirmed in vitro by native gel electrophoresis and size exclusion chromatography. Determination of the steady-state kinetics of tRNA Pro charging showed that the catalytic efficiency (k cat /K m ) of ProRS increased 5-fold in the complex with LeuRS compared with the free enzyme, whereas the K m for proline was unchanged. No significant changes in the steady-state kinetics of LeuRS aminoacylation were observed upon the addition of ProRS. These findings indicate that ProRS and LeuRS associate in M. thermautotrophicus and suggest that this interaction contributes to translational fidelity by enhancing tRNA aminoacylation by ProRS.Aminoacyl-tRNA synthetases (aaRSs) 1 are essential components of the translation process. Their cellular role is to ensure that individual tRNAs are attached to their cognate amino acid. Each aaRS specifically binds to a defined set of tRNAs and catalyzes the attachment of the amino acids to the tRNAs. Once synthesized, the aminoacyl-tRNAs function as substrates for ribosomal protein synthesis, thereby ensuring correct translation of the genetic code (1). In bacteria, aaRSs typically perform their role as individual enzymes, found either as monomers, homodimers, or homo-or heterotetramers. However, in eukaryotes several aminoacyl-tRNA synthetases exist in multienzyme complexes (2-4), and two different types have so far been found in mammalian cells. One is composed of only one aminoacyl-tRNA synthetase, valyl-tRNA synthetase, and EF-1H, the heavy form of translation elongation factor 1 (5). The complex is believed to contain seven subunits, two monomeric subunits of valyl-tRNA synthetase and the EF-1H subunits EF1-␣, -␤, -␥, and -␦ in the molar ratio 2:1:1:1. The second complex described is considerably larger and includes nine aminoacyl-tRNA synthetase activities. The complex is composed of isoleucyl-, leucyl-(LeuRS), prolyl-(ProRS), methionyl-, glutaminyl-, glutamyl-, lysyl-, arginyl-, and aspartyl-tRNA synthetases. Whereas most of the aaRSs are present in the complex as monomers, data have indicated that lysyltRNA synthetase and aspartyl-tRNA synthetase exist as dimers (3,6). In addition, the polypeptide carrying the ProRS activity is multifunctional in that the protein also comprises the catalytic domain and activity of glutamyl-tRNA synthetase (7). Three auxiliary proteins, p18, p38, and p43, are also part of the multisynthetase complex. Although the structural and functional significance of the complex still remains to be elu...

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“…1). Truncation of the N-terminal extension decreased the catalytic efficiency only by threefold as a result of the reduced affinity for tRNA Lys and had no effect on the interaction with p38 (10,17,18). Both the full-length LysRS (M1-V597) and truncation (S70-T584) that removed the eukaryote-specific extension were expressed, purified, and attempted for crystallization.…”

Section: General Features Of Lysrss In Evolutionmentioning

confidence: 99%

Crystal structure of tetrameric form of human lysyl-tRNA synthetase: Implications for multisynthetase complex formation

Guo

Ignatov

Musier‐Forsyth

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

114

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In mammals, many aminoacyl-tRNA synthetases are bound together in a multisynthetase complex (MSC) as a reservoir of procytokines and regulation molecules for functions beyond aminoacylation. The ␣2 homodimeric lysyl-tRNA synthetase (LysRS) is tightly bound in the MSC and, under specific conditions, is secreted to trigger a proinflammatory response. Results by others suggest that ␣2 LysRS is tightly bound into the core of the MSC with homodimeric ␤2 p38, a scaffolding protein that itself is multifunctional. Not understood is how the two dimeric proteins combine to make a presumptive ␣2␤2 heterotetramer and, in particular, the location of the surfaces on LysRS that would accommodate the p38 interactions. Here we present a 2.3-Å crystal structure of a tetrameric form of human LysRS. The relatively loose (as seen in solution) tetramer interface is assembled from two eukaryotespecific sequences, one in the catalytic-and another in the anticodon-binding domain. This same interface is predicted to provide unique determinants for interaction with p38. The analyses suggest how the core of the MSC is assembled and, more generally, that interactions and functions of synthetases can be built and regulated through dynamic protein-protein interfaces. These interfaces are created from small adaptations to what is otherwise a highly conserved (through evolution) polypeptide sequence.AIMP2 ͉ aminoacyl-tRNA synthetase ͉ p38

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The N-terminal Domain of Mammalian Lysyl-tRNA Synthetase Is a Functional tRNA-binding Domain

Cited by 103 publications

References 51 publications

A glycolytic enzyme, enolase, is recruited as a cofactor of tRNA targeting toward mitochondria in Saccharomyces cerevisiae

A glycolytic enzyme, enolase, is recruited as a cofactor of tRNA targeting toward mitochondria in Saccharomyces cerevisiae

Association between Archaeal Prolyl- and Leucyl-tRNA Synthetases Enhances tRNAPro Aminoacylation

Crystal structure of tetrameric form of human lysyl-tRNA synthetase: Implications for multisynthetase complex formation

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