Isolation and characterization of human nuclear and cytosolic multisynthetase complexes and the intracellular distribution of p43/EMAPII.
In this study, the human multienzyme aminoacyl-tRNA synthetase "core" complex has been isolated from the nuclear and cytosolic compartments of human cells and purified to near homogeneity. It is clear from the polypeptide compositions, stoichiometries, and three-dimensional structures that the cytosolic and nuclear particles are very similar to each other and to the particle obtained from rabbit reticulocytes. The most significant difference observed via aminoacylation activity assays and densitometric analysis of electrophoretic band patterns is a lower amount of glutaminyl-tRNA synthetase in the human particles. However, this is not enough to cause major changes in the three-dimensional structures calculated from samples negatively stained with either uranyl acetate or methylamine vanadate. Indeed, the latter samples produce volumes that are highly similar to an initial structure previously calculated from a frozen hydrated sample of the rabbit multisynthetase complex. New structures in this study reveal that the three major structural domains have discrete subsections. This information is an important step toward determination of specific protein interactions and arrangements within the multisynthetase core complex and understanding of the particle's cellular function(s). Finally, gel filtration and immunoblot analysis demonstrate that a major biological role for the cytokine precursor p43 is as an integral part of the multisynthetase complex.
D000596:Amino Acids / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D000818:Animals; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D016207:Cytokines / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005975:Glutamate-tRNA Ligase / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D015151:Immunoblotting; D020014:K562 Cells / Q000737:chemistry / Q000201:enzymology; D007700:Kinetics; D008854:Microscopy, Electron; D008958:Models, Molecular; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D040681:Structural Homology, Protein
D000596:Amino Acids; D016207:Cytokines; D009363:Neoplasm Proteins; D016601:RNA-Binding Proteins; C077219:small inducible cytokine subfamily E, member 1; D000604:Amino Acyl-tRNA Synthetases; D005975:Glutamate-tRNA Ligase