Nuclear Localization of Aminoacyl-tRNA Synthetases Using Single-Cell Capillary Electrophoresis Laser-Induced Fluorescence Analysis
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- 作者：Nilhan Gunasekera ; Sang Won Lee ; Sunghoon Kim ; Karin Musier-Forsyth ; and Edgar Arriaga
- 刊名：Analytical Chemistry
- 出版年：2004
- 出版时间：August 15, 2004
- 年：2004
- 卷：76
- 期：16
- 页码：4741 - 4746
- 全文大小：91K
- 年卷期：v.76,no.16(August 15, 2004)
- ISSN：1520-6882
文摘
Aminoacyl-tRNA synthetases (aaRSs) are a family ofenzymes whose function in specific aminoacylation oftRNAs is central to the process of protein translation,which occurs in the cytoplasm of all living cells. Inaddition to their well-established cytoplasmic localization,fluorescence microscopy studies and analysis of theaminoacylation state of nuclear tRNAs have revealed thatsynthetases are localized in the nuclei of cells from severalspecies including Xenopus laevis and Saccharomycescerevisiae. Whether nuclear localization of aaRSs is ageneral phenomenon that occurs in all eukaryotic cells isan open question. In the work described here, humanmethionyl-tRNA synthetase (MRS) and human lysyl-tRNAsynthetase (KRS) were expressed in human-derived 
ta.gif" BORDER=0 >H2-1osteosarcoma cells as enhanced green fluorescent protein(EGFP) fusion proteins. The subcellular localization ofthese EGFP-aaRSs was first probed by fluorescencemicroscopy using cells that coexpressed EGFP-aaRS anda 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 confirmthese findings, and to investigate a potentially moresensitive, general method for nuclear localization studies,capillary electrophoresis with laser-induced fluorescence(CE-LIF) detection was used to analyze single ta.gif" BORDER=0 >H2-1 cellsexpressing both EGFP-aaRS and nuDsRed. While cytosolic EGFP signals were detected for both EGFP-MRS andEGFP-KRS, only EGFP-MRS was found in the nucleus,along with nuDsRed. The detection of EGFP-MRS innuclei of ta.gif" BORDER=0 >H2-1 cells demonstrates the feasibility of usingCE-LIF analysis in nuclear localization studies of proteinsin mammalian cells.
ta.gif" BORDER=0 >H2-1osteosarcoma cells as enhanced green fluorescent protein(EGFP) fusion proteins. The subcellular localization ofthese EGFP-aaRSs was first probed by fluorescencemicroscopy using cells that coexpressed EGFP-aaRS anda 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 confirmthese findings, and to investigate a potentially moresensitive, general method for nuclear localization studies,capillary electrophoresis with laser-induced fluorescence(CE-LIF) detection was used to analyze single ta.gif" BORDER=0 >H2-1 cellsexpressing both EGFP-aaRS and nuDsRed. While cytosolic EGFP signals were detected for both EGFP-MRS andEGFP-KRS, only EGFP-MRS was found in the nucleus,along with nuDsRed. The detection of EGFP-MRS innuclei of ta.gif" BORDER=0 >H2-1 cells demonstrates the feasibility of usingCE-LIF analysis in nuclear localization studies of proteinsin mammalian cells.