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Active Aminoacyl-tRNA Synthetases Are Present in Nuclei as a High Molecular Weight Multienzyme Complex*

  • Lubov Nathanson
    Affiliations
    Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33136
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  • Murray P. Deutscher
    Correspondence
    To whom correspondence should be addressed. Tel.: 305-243-3150; Fax: 305-243-3955
    Affiliations
    Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33136
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  • Author Footnotes
    * This work was supported by Grant GM16317 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:October 13, 2000DOI:https://doi.org/10.1074/jbc.C000385200
      Recent studies suggest that aminoacylation of tRNA may play an important role in the transport of these molecules from the nucleus to the cytoplasm. However, there is almost no information regarding the status of active aminoacyl-tRNA synthetases within the nuclei of eukaryotic cells. Here we show that at least 13 active aminoacyl-tRNA synthetases are present in purified nuclei of both Chinese hamster ovary and rabbit kidney cells, although their steady-state levels represent only a small percentage of those found in the cytoplasm. Most interestingly, all the nuclear aminoacyl-tRNA synthetases examined can be isolated as part of a multienzyme complex that is more stable, and consequently larger, than the comparable complex isolated from the cytoplasm. These data directly demonstrate the presence of active aminoacyl-tRNA synthetases in mammalian cell nuclei. Moreover, their unexpected structural organization raises important questions about the functional significance of these multienzyme complexes and whether they might play a more direct role in nuclear to cytoplasmic transport of tRNAs.
      EF1
      elongation factor 1
      CHO
      Chinese hamster ovary
      PBS
      phosphate-buffered saline
      Aminoacyl-tRNA synthetases catalyze the first step in protein biosynthesis, the attachment of an amino acid to its cognate tRNA (
      • Ibba M.
      • Curnow A.W.
      • Söll D.
      ). In higher eukaryotes the aminoacyl-tRNA synthetases are part of a highly organized translation system (
      • Negrutskii B.S.
      • Stapulionis R.
      • Deutscher M.P.
      ,
      • Stapulionis R.
      • Deutscher M.P.
      ) and can be isolated from cells as a high molecular weight multienzyme complex (
      • Bandyopadhyay A.K.
      • Deutscher M.P.
      ,
      • Mirande M.
      ,
      • Kisselev L.L.
      • Wolfson A.D.
      ,
      • Yang D.C.H.
      ). The number of synthetases in this fragile complex varies in different laboratories, but a stable core of nine of these enzymes plus three non-synthetase proteins can be isolated reproducibly (
      • Mirande M.
      ,
      • Kisselev L.L.
      • Wolfson A.D.
      ,
      • Yang D.C.H.
      ). Evidence has accumulated that the multienzyme synthetase complex reflects associations among these proteins that pre-exist in vivo(
      • Filonenko V.V.
      • Deutscher M.P.
      ,
      • Quevillon S.
      • Robinson J.-C.
      • Berthonneau E.
      • Siatecka M.
      • Mirande M.
      ,
      • Rho S.B.
      • Kim M.J.
      • Lee J.S.
      • Seol W.
      • Motegi H.
      • Kim S.
      • Shiba K.
      ).
      Despite the fact that protein synthesis takes place in the cytoplasm of eukaryotic cells, several studies suggested that aminoacyl-tRNA synthetases might also be present in nuclei. Thus, in early work, aminoacyl-tRNA synthetase activities could be detected in crude nuclear fractions (
      • Amar-Costesec A.
      • Turu C.
      ,
      • Hampel A.
      • Enger M.D.
      ); however, it could be argued that these activities were due to cytoplasmic adherence to the outer nuclear surface. Aminoacyl-tRNA synthetases, as well as elongation factor 1 (EF1),1 also could be detected in nuclei by immunochemical methods (
      • Mirande M.
      • LeCorre D.
      • Waller J.-P.
      ,
      • Popenko V.I.
      • Ivanova J.L.
      • Cherny N.E.
      • Filonenko V.V.
      • Beresten S.F.
      • Wolfson A.D.
      • Kisselev L.L.
      ,
      • Barbarase E.
      • Koppel D.E.
      • Deutscher M.P.
      • Smith C.L.
      • Ainger K.
      • Morgan F.
      • Carson J.H.
      ,
      • Sanders J.
      • Brandsma M.
      • Janssen G.M.C.
      • Dijk J.
      • Möller V.
      ). However, these studies provided no information as to whether the nuclear-localized synthetases were active. Nuclear localization was also suggested by the identification of possible nuclear localization signals in yeast aminoacyl-tRNA synthetases (
      • Schimmel P.
      • Wang C.-C.
      ).
      Recently, nuclear aminoacyl-tRNA synthetases have attracted considerable interest because of their potential involvement in nuclear to cytoplasmic transport of tRNA. Exclusion of defective tRNA from the cytoplasm was shown to be due to nuclear proofreading, and it was proposed that aminoacylation of tRNA serves as this proofreading step (
      • Lund E.
      • Dahlberg J.E.
      ,
      • Sarkar S.
      • Azad A.K.
      • Hopper A.K.
      ,
      • Hopper A.K.
      ). While there is still some discussion whether aminoacylation or binding to the nuclear export receptor, exportin-t, is actually responsible for proofreading, there is agreement that nuclear aminoacylation increases tRNA export efficiency (
      • Arts G.-J.
      • Kuersten S.
      • Romby P.
      • Ehresmann B.
      • Mattaj I.W.
      ,
      • Lipowsky G.
      • Bischoff F.R.
      • Tzaurralde E.
      • Kutay U.
      • Schäfer S.
      • Gross H.J.
      • Beier H.
      • Görlich D.
      ).
      In light of these findings, it was of interest to carefully examine nuclei for the presence of active aminoacyl-tRNA synthetases and to determine what percentage of total cellular synthetase activity might reside in the nucleus. The data reported here directly demonstrate that active aminoacyl-tRNA synthetases are present in the nuclei of two different mammalian cell lines. Most importantly, we find that the nuclear aminoacyl-tRNA synthetases are organized into a high molecular weight, multienzyme complex that is even more stable than the complex present in the cytoplasm.

      Acknowledgement

      We thank Olena Shcherbyna-Hawks for excellent technical assistance.

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