Nuclear localization of aminoacyl-tRNA synthetases using single-cell capillary electrophoresis laser-induced fluorescence analysis.
Aminoacyl-tRNA synthetases (aaRSs) are a family of enzymes whose function in specific aminoacylation of tRNAs is central to the process of protein translation, which occurs in the cytoplasm of all living cells. In addition to their well-established cytoplasmic localization, fluorescence microscopy studies and analysis of the aminoacylation state of nuclear tRNAs have revealed that synthetases are localized in the nuclei of cells from several species including Xenopus laevis and Saccharomyces cerevisiae. Whether nuclear localization of aaRSs is a general phenomenon that occurs in all eukaryotic cells is an open question. In the work described here, human methionyl-tRNA synthetase (MRS) and human lysyl-tRNA synthetase (KRS) were expressed in human-derived DeltaH2-1 osteosarcoma cells as enhanced green fluorescent protein (EGFP) fusion proteins. The subcellular localization of these EGFP-aaRSs was first probed by fluorescence microscopy using cells that coexpressed EGFP-aaRS and a nuclear marker fusion protein, nuDsRed. As expected, both aaRSs were present in the cytosol, while only EGFP-MRS was also clearly localized in the nucleus. To confirm these findings, and to investigate a potentially more sensitive, general method for nuclear localization studies, capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was used to analyze single DeltaH2-1 cells expressing both EGFP-aaRS and nuDsRed. While cytosolic EGFP signals were detected for both EGFP-MRS and EGFP-KRS, only EGFP-MRS was found in the nucleus, along with nuDsRed. The detection of EGFP-MRS in nuclei of DeltaH2-1 cells demonstrates the feasibility of using CE-LIF analysis in nuclear localization studies of proteins in mammalian cells.
Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455, USA.
Nilhan
N
Gunasekera
Sang Won
SW
Lee
Sunghoon
S
Kim
Karin
K
Musier-Forsyth
Edgar
E
Arriaga
D000604:Amino Acyl-tRNA Synthetases / Q000302:isolation & purification*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000201:enzymology*; D019075:Electrophoresis, Capillary / Q000379:methods; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D007834:Lasers; D013050:Spectrometry, Fluorescence / Q000379:methods
D049452:Green Fluorescent Proteins; D000604:Amino Acyl-tRNA Synthetases