Genetic dissection of protein–protein interactions in multi-tRNA synthetase complex

Rho, Seung Bae; Kim, Min Jung; Lee, Jong Sang; Seol, Wongi; Motegi, Hiromi; Kim, Sung Hoon; Shiba, Kiyotaka

doi:10.1073/pnas.96.8.4488

Cited by 121 publications

(133 citation statements)

References 57 publications

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“…However, by combining the information in TABLE ONE, previous results, and the new data, it is possible to make some reasonable predictions. For example, interactions between LeuRS and GluProRS have been demonstrated by chemical cross-linking (14) and genetic analyses (15,16). Together with IleRS, these same two enzymes form a distinct sub-complex of the multisynthetase particle (13).…”

Section: Resultsmentioning

confidence: 99%

“…The supporting data were from cross-linking reactions (14) as well as genetic two-hybrid studies (7,16). Spot 3 is on the midline of the vertical axis of the particle.…”

Section: Isolation Of Multisynthetase Complex Via a Novel Methods And mentioning

confidence: 99%

“…Then, a number of nearest-neighbor protein interactions within the isolated complex were detected via chemical cross-linking studies (14). Using an in vivo approach, a number of potential component interactions have been identified by yeast two-hybrid genetic screens (15,16). Surface plasmon resonance measurements were used to examine interactions of individual proteins from the complex (8).…”

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confidence: 99%

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A Three-dimensional Working Model of the Multienzyme Complex of Aminoacyl-tRNA Synthetases Based on Electron Microscopic Placements of tRNA and Proteins

Wolfe

Warrington

Treadwell

et al. 2005

Journal of Biological Chemistry

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It has become evident that the process of protein synthesis is performed by many cellular polypeptides acting in concert within the structural confines of protein complexes. In multicellular eukaryotes, one of these assemblies is a multienzyme complex composed of eight proteins that have aminoacyl-tRNA synthetase activities as well as three non-synthetase proteins (p43, p38, and p18) with diverse functions. This study uses electron microscopy and three-dimensional reconstruction to explore the arrangement of proteins and tRNA substrates within this "core" multisynthetase complex. Binding of unfractionated tRNA establishes that these molecules are widely distributed on the exterior of the structure. Binding of gold-labeled tRNALeu places leucyl-tRNA synthetase and the bifunctional glutamyl-/prolyl-tRNA synthetase at the base of this asymmetric "V"-shaped particle. A stable cell line has been produced that incorporates hexahistidine-labeled p43 into the multisynthetase complex. Using a gold-labeled nickel-nitrilotriacetic acid probe, the polypeptides of the p43 dimer have been located along one face of the particle. The results of this and previous studies are combined into an initial three-dimensional working model of the multisynthetase complex. This is the first conceptualization of how the protein constituents and tRNA substrates are arrayed within the structural confines of this multiprotein assembly.It has been known for many years that a "core" complex of aminoacyltRNA synthetases can be isolated from all multicellular eukaryotes (1). Its characteristic composition is three non-synthetase proteins and eight polypeptides having nine aminoacyl-tRNA synthetase activities. With the enzymes abbreviated as the three-letter amino acid name plus RS for the enzyme, the components are ArgRS, AspRS, GluProRS, GlnRS, IleRS, LeuRS, LysRS, MetRS, p43, p38, and p18. Several of these are known dimers, so the total polypeptide count in the multisynthetase complex is at least fifteen. This is a particularly intriguing protein assembly as all of the enzymes catalyze the same reaction, albeit with individual and highly specific substrates. The reaction catalyzed is the covalent attachment of an amino acid to either the 2Ј-or 3Ј-hydroxyl of the 3Ј-terminal adenosine of tRNA. Recent increased interest in these enzymes has been generated by studies indicating that they have roles in a variety of other cellular processes (2-4). Such activities range from cytokine-mediated chemoattraction to priming of reverse transcription of the human immunodeficiency virus, type 1 genome. These enzymes are also viewed as targets in the development of new antimicrobial agents (5). The multisynthetase complex also contains three non-synthetase proteins. The first of these, p18, has been shown to interact with EF-1␥, which may facilitate transfer of aminoacyl-tRNAs to the ribosome (6). Data from yeast two-hybrid screens (7), in vitro binding assays (8), and deletion analysis (9) indicate that p38 functions as a scaffolding protein and appears to be...

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Section: Resultsmentioning

confidence: 99%

“…The supporting data were from cross-linking reactions (14) as well as genetic two-hybrid studies (7,16). Spot 3 is on the midline of the vertical axis of the particle.…”

Section: Isolation Of Multisynthetase Complex Via a Novel Methods And mentioning

confidence: 99%

mentioning

confidence: 99%

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A Three-dimensional Working Model of the Multienzyme Complex of Aminoacyl-tRNA Synthetases Based on Electron Microscopic Placements of tRNA and Proteins

Wolfe

Warrington

Treadwell

et al. 2005

Journal of Biological Chemistry

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“…Furthermore, intact rat aspartyl-tRNA synthetase (DRS) associates in vivo with the multi-ARS complex, whereas the Nterminal appendix-deleted form does not (5), indicating that the appended domain is indispensable for targeting DRS to this complex. Protein-protein interactions between the appendices of various ARSs have previously been shown using the yeast two-hybrid system (6). Similarly, p43 associates with the Nterminal appendix of human arginyl-tRNA synthetase (RRS), stimulating its aminoacylation activity (7).…”

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confidence: 99%

Catalytic Peptide of Human Glutaminyl-tRNA Synthetase Is Essential for Its Assembly to the Aminoacyl-tRNA Synthetase Complex

Kim¹,

Park²,

Kim³

et al. 2000

Journal of Biological Chemistry

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Human glutaminyl-tRNA synthetase (QRS) is one of several mammalian aminoacyl-tRNA synthetases (ARSs) that form a macromolecular protein complex. To understand the mechanism of QRS targeting to the multi-ARS complex, we analyzed both exogenous and endogenous QRSs by immunoprecipitation after overexpression of various Myc-tagged QRS mutants in human embryonic kidney 293 cells. Whereas a deletion mutant containing only the catalytic domain (QRS-C) was targeted to the multi-ARS complex, a mutant QRS containing only the N-terminal appended domain (QRS-N) was not. Deletion mapping showed that the ATP-binding Rossman fold was necessary for targeting of QRS to the multi-ARS complex. Furthermore, exogenous Myc-tagged QRS-C was co-immunoprecipitated with endogenous QRS. Since glutaminylation of tRNA was dramatically increased in cells transfected with the full-length QRS, but not with either QRS-C or QRS-N, both the QRS catalytic domain and the N-terminal appended domain were required for full aminoacylation activity. When QRS-C was overexpressed, arginyl-tRNA synthetase and p43 were released from the multi-ARS complex along with endogenous QRS, suggesting that the N-terminal appendix of QRS is required to keep arginyl-tRNA synthetase and p43 within the complex. Thus, the eukaryote-specific N-terminal appendix of QRS appears to stabilize the association of other components in the multi-ARS complex, whereas the C-terminal catalytic domain is necessary for QRS association with the multi-ARS complex.Aminoacyl-tRNA synthetases (ARSs) 1 catalyze the ligation of a specific amino acid to its cognate tRNA, thereby ensuring the faithful translation of genetic code. Although catalytic domains from homologous synthetases are highly conserved from bacteria to mammals, mammalian ARSs have acquired unique peptide appendices that are not present in the corresponding prokaryotic enzymes (1-3). Eight of the mammalian ARSs are found in a high molecular weight multi-ARS complex with three noncatalytic proteins, p43, p38, and p18, whereas the corresponding bacterial ARSs do not form such a complex (1-4). This suggests that the peptide appendices might be involved in protein-protein interactions within the multi-ARS complex. Furthermore, intact rat aspartyl-tRNA synthetase (DRS) associates in vivo with the multi-ARS complex, whereas the Nterminal appendix-deleted form does not (5), indicating that the appended domain is indispensable for targeting DRS to this complex. Protein-protein interactions between the appendices of various ARSs have previously been shown using the yeast two-hybrid system (6). Similarly, p43 associates with the Nterminal appendix of human arginyl-tRNA synthetase (RRS), stimulating its aminoacylation activity (7).In addition to a role in protein-protein interactions, ARS appended domains could also enhance aminoacylation activity by recruiting tRNAs. Previously it has been shown that the N-terminal appended domain of yeast QRS nonspecifically binds to both double-and single-stranded RNA (8). Fusion of this domain to Esch...

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“…The assembly of the complex is mediated by heat-shock protein 90 (4) and involves protein-protein interactions via the unique noncatalytic peptides attached to each of the component enzymes (5)(6)(7) and their catalytic core domains (8). It was expected that the three associating factors would also contribute to the complex formation.…”

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confidence: 99%

p38 is essential for the assembly and stability of macromolecular tRNA synthetase complex: Implications for its physiological significance

Kim

Kang

Lee

et al. 2002

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

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113

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Mammalian tRNA synthetases form a macromolecular complex with three nonenzyme factors: p43, p38, and p18. Here we introduced a mutation within the mouse p38 gene to understand its functional significance for the formation of the multi-tRNA synthetase complex. The complex was completely disintegrated by the deficiency of p38. In addition, the protein levels and catalytic activities of the component enzymes and cofactors were severely decreased. A partial truncation of the p38 polypeptide separated the associated components into different subdomains. The mutant mice showed lethality within 2 days of birth. Thus, this work provides the first evidence, to our knowledge, that p38 is essential for the structural integrity of the multi-tRNA synthetase complex and mouse viability.aminoacyl-tRNA synthetase ͉ macromolecular protein complex ͉ gene trap ͉ protein-protein interaction A minoacyl-tRNA synthetases (ARSs) are essential enzymes catalyzing the ligation of their cognate amino acids and tRNAs. Eight different enzymes of higher eukaryotes form a macromolecular complex with three nonsynthetase factors: p43, p38, and p18 (1-3). Although this complex was first reported more than two decades ago, the functional reason for their molecular assembly and the structural organization of the components still remain unknown.Recently, much information has been obtained about the associations and interactions of the component proteins. The assembly of the complex is mediated by heat-shock protein 90 (4) and involves protein-protein interactions via the unique noncatalytic peptides attached to each of the component enzymes (5-7) and their catalytic core domains (8). It was expected that the three associating factors would also contribute to the complex formation. Among the three auxiliary factors, the interaction and function of p43 have been best elucidated. The p43 protein is located in the middle of the complex (9) and is associated with arginyl-tRNA synthetase via its N-terminal region (10). Its C-terminal domain contains an OB-fold, which is responsible for the interaction with tRNA (11, 12) and facilitates the catalytic activity of the bound enzyme (10). Interestingly, p43 is also secreted to work as a proinflammatory cytokine (13,14). The functions of the two other factors are less understood. p38 interacts with many components of the complex (2, 15). To understand the in vivo functional significance of p38 in the formation of the multi-ARS complex, we mutated the p38 structural gene in the mouse and investigated its effects on the cellular stability of the multi-ARS complex and the component proteins. Deletion analyses of p38 were also conducted to map the organization of the component proteins within the complex.

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Genetic dissection of protein–protein interactions in multi-tRNA synthetase complex

Cited by 121 publications

References 57 publications

A Three-dimensional Working Model of the Multienzyme Complex of Aminoacyl-tRNA Synthetases Based on Electron Microscopic Placements of tRNA and Proteins

A Three-dimensional Working Model of the Multienzyme Complex of Aminoacyl-tRNA Synthetases Based on Electron Microscopic Placements of tRNA and Proteins

Catalytic Peptide of Human Glutaminyl-tRNA Synthetase Is Essential for Its Assembly to the Aminoacyl-tRNA Synthetase Complex

p38 is essential for the assembly and stability of macromolecular tRNA synthetase complex: Implications for its physiological significance

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