Engineering mammalian aspartyl-tRNA synthetase to probe structural features mediating its association with the multisynthetase complex.
Aspartyl-tRNA synthetase from higher eukaryotes is a component of a multienzyme complex comprising nine aminoacyl-tRNA synthetases. The cDNA encoding cytoplasmic rat liver aspartyl-tRNA synthetase was previously cloned and sequenced. This work reports the identification of structural features responsible for its association within the multisynthetase complex. Mutant and chimeric proteins have been expressed in mammalian cells and their structural behavior analyzed. A wild-type rat liver aspartyl-tRNA synthetase, expressed in Chinese hamster ovary (CHO) cells, associates within the complex from CHO cells, whereas a mutant enzyme with a deletion of 34 amino acids from its amino-terminal extremity does not. A chimeric enzyme, made of the amino-terminal moiety of rat liver aspartyl-tRNA synthetase fused to the catalytic domain of yeast lysyl-tRNA synthetase, has been expressed in Lys-101 cells, a CHO cell line with a temperature-sensitive lysyl-tRNA synthetase. The fusion protein is stable in vivo, does not associate within the multisynthetase complex and cannot restore normal growth of the mutant cells. These results establish that the 3.7-kDa amino-terminal moiety of mammalian aspartyl-tRNA synthetase mediates its association with the other components of the complex. In addition, the finding that yeast lysyl-tRNA synthetase cannot replace the aspartyl-tRNA synthetase component of the mammalian complex, indicates that interactions between neighbouring enzymes also play a prominent role in stabilization of this multienzyme structure and strengthened the view that the multisynthetase complex is a discrete entity with a well-defined structural organization.
Mirande
M
M
Laboratoire d'Enzymologie du Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
Lazard
M
M
Martinez
R
R
Latreille
M T
MT
D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics; D000818:Animals; D001226:Aspartate-tRNA Ligase / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D002850:Chromatography, Gel; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D051381:Rats; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D009097:Multienzyme Complexes; D004247:DNA; D000604:Amino Acyl-tRNA Synthetases; D001226:Aspartate-tRNA Ligase