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Eukaryotic Cell, October 2008, p. 1819-1830, Vol. 7, No. 10
1535-9778/08/$08.00+0     doi:10.1128/EC.00088-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

TOR1 and TOR2 Have Distinct Locations in Live Cells{triangledown} ,{dagger}

Thomas W. Sturgill,1* Adiel Cohen,2 Melanie Diefenbacher,2 Mark Trautwein,2 Dietmar E. Martin,2 and Michael N. Hall2

Department of Pharmacology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908,1 Biozentrum, University of Basel, CH-4056 Basel, Switzerland2

Received 11 March 2008/ Accepted 9 July 2008

TOR is a structurally and functionally conserved Ser/Thr kinase found in two multiprotein complexes that regulate many cellular processes to control cell growth. Although extensively studied, the localization of TOR is still ambiguous, possibly because endogenous TOR in live cells has not been examined. Here, we examined the localization of green fluorescent protein (GFP) tagged, endogenous TOR1 and TOR2 in live S. cerevisiae cells. A DNA cassette encoding three copies of green fluorescent protein (3XGFP) was inserted in the TOR1 gene (at codon D330) or the TOR2 gene (at codon N321). The TORs were tagged internally because TOR1 or TOR2 tagged at the N or C terminus was not functional. The TOR1D330-3XGFP strain was not hypersensitive to rapamycin, was not cold sensitive, and was not resistant to manganese toxicity caused by the loss of Pmr1, all indications that TOR1-3XGFP was expressed and functional. TOR2-3XGFP was functional, as TOR2 is an essential gene and TOR2N321-3XGFP haploid cells were viable. Thus, TOR1 and TOR2 retain function after the insertion of 748 amino acids in a variable region of their noncatalytic domain. The localization patterns of TOR1-3XGFP and TOR2-3XGFP were documented by imaging of live cells. TOR1-3XGFP was diffusely cytoplasmic and concentrated near the vacuolar membrane. The TOR2-3XGFP signal was cytoplasmic but predominately in dots at the plasma membrane. Thus, TOR1 and TOR2 have distinct localization patterns, consistent with the regulation of cellular processes as part of two different complexes.

* Corresponding author. Mailing address: Department of Pharmacology, Box 800735, Room 5045, Jordan Hall, University of Virginia Health Sciences Center, Charlottesville, VA 22908. Phone: (434) 924-1919. Fax: (434) 924-5207. E-mail: Thomas_Sturgill{at}virginia.edu

{triangledown} Published ahead of print on 22 August 2008.

{dagger} Supplemental material for this article may be found at http://ec.asm.org/.

Eukaryotic Cell, October 2008, p. 1819-1830, Vol. 7, No. 10
1535-9778/08/$08.00+0     doi:10.1128/EC.00088-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Schonbrun, M., Laor, D., Lopez-Maury, L., Bahler, J., Kupiec, M., Weisman, R. (2009). TOR Complex 2 Controls Gene Silencing, Telomere Length Maintenance, and Survival under DNA-Damaging Conditions. Mol. Cell. Biol. 29: 4584-4594 [Abstract] [Full Text]  
  • Singh, J., Tyers, M. (2009). A Rab escort protein integrates the secretion system with TOR signaling and ribosome biogenesis. Genes Dev. 23: 1944-1958 [Abstract] [Full Text]  


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