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. 2008 Oct;7(10):1819-30.
doi: 10.1128/EC.00088-08. Epub 2008 Aug 22.

TOR1 and TOR2 have distinct locations in live cells

Thomas W Sturgill  1 , Adiel Cohen, Melanie Diefenbacher, Mark Trautwein, Dietmar E Martin, Michael N Hall
Affiliations

TOR1 and TOR2 have distinct locations in live cells

Thomas W Sturgill et al. Eukaryot Cell. 2008 Oct.

Abstract

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 TOR1(D330-3XGFP) 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 TOR2(N321-3XGFP) 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.

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Figures

FIG. 1.
FIG. 1.
The alignment of evolutionary divergent species identifies a region of TOR for the insertion of 3XGFP. Proteins were aligned by ClustalW (7). Only the relevant portion is shown. Residues D67 and D330 in TOR1 and N321 in TOR2 are indicated by asterisks. Arrows indicate sites where 3XGFP was inserted at D330 (TOR1) or N321 (TOR2) with retention of function. Humans, Caenorhabditis elegans, and Cryptococcus neoformans have only one TOR. Default colors and alignment parameters are from the implementation of ClustalW (7) at http://npsa-pbil.ibcp.fr/.
FIG. 2.
FIG. 2.
TOR2-3XGFP is expressed, and TOR1D330-3XGFP strains have normal sensitivity to rapamycin. (A) Western blot with anti-GFP antibodies of 40 μg of total protein (see Materials and Methods) of the control (Ctl), TB50a, and VA34 strains. The indicated strains were streaked onto YPD (B), YPD plus 1 nM rapamycin (Rap) (C), and YPD plus 2 nM rapamycin (D). Cells were grown for 2 days at 30°C after streaking and then scanned.
FIG. 3.
FIG. 3.
TOR1D330-3XGFP strains adapt to cold stress. The indicated strains were streaked onto YPD (A) or YPD plus 2 nM rapamycin (Rap) (B) Cells were grown for 4 days at 15°C (15 deg C) after streaking and then scanned.
FIG. 4.
FIG. 4.
TOR1D330-3XGFP, like TOR1, is sensitive to Mn2+ in a pmr1Δ strain. The indicated strains were streaked onto YPD (A) or YPD containing 2 mM Mn2+ (final concentration) (B). Cells were grown for 2 days at 30°C, and then plates were scanned. The TOR1 PMR1 (TB50a), tor1Δ PMR1 (AN9-2a), tor1Δ pmr1Δ (YGD25), TOR1 pmr1Δ (LJ25-1A), TOR1D330-3XGFP pmr1Δ (VA68-9c), and TOR1D330-3XGFP PMR1 (VA34) strains were used (Table 3).
FIG. 5.
FIG. 5.
TOR1D67-3XGFP is partially functional. (A) Growth on YPD containing 1 nM rapamycin. The indicated strains (Table 3) were streaked, and after 3 days of growth, the plate was scanned. Growth patterns on YPD from streaks in the same experiment were similar (data not shown). (B) Growth at 15°C (15 deg) on YPD or YPD plus 1 nM rapamycin (Rap). The indicated strains were assayed by a 10-fold dilution and pinning; TOR1D67-3XGFP (VA38) was used. The plates were scanned after 4 days.
FIG. 6.
FIG. 6.
TOR1D330-3XGFP is predominantly cytoplasmic and concentrated as dots near the vacuolar membrane. (A) Localization of TOR1-3XGFP in cells grown in YDP. Merged GFP (green; GFP channel) and autofluorescence images for TB50a (control) lacking a GFP cassette and TOR1-3XGFP (VA34) strains are shown. Strains were grown overnight in YPD, diluted, and imaged while still in log phase after centrifugation and suspension in synthetic medium. The arrow indicates a dot near the vacuole, and the feathered arrow indicates a dot near the vacuolar membrane. (B) The TOR1-3XGFP signal overlaps FM4-64 staining (see Materials and Methods) of the vacuolar membrane. Control, strain TB50a lacking a GFP cassette; TOR1-3XGFP, strain VA102. The exposure settings used were as follows: DIC, 300 ms; GFP, 10,000 ms; and Fm4-64 (Cy3), 10,000 ms.
FIG. 7.
FIG. 7.
TOR1D330-3XGFP localization is distinct from that of Sec7-dsRed and similar to that of FYVE-dsRed. We performed microscopy with the following strains in SD or SD-leu (top to bottom): TB50a (control strain, in SD), TOR1D330-3XGFP (V66, in SD), TOR1D330-3XGFP plus FYVE-dsRed (VA66 transformed with pTPQ127, in SD-leu), and TOR1D330-3XGFP plus Sec7-dsRed (VA66 transformed with pTPQ128, in SD-leu). The arrow shows an example of colocalization of TOR1D330-3XGFP with an FYVE domain marker in a punctate structure. The exposure settings used were as follows: DIC, 100 ms; GFP, 2,000 ms; and dsRed, 500 ms.
FIG. 8.
FIG. 8.
TOR2 is functional if N321 is replaced by 1X-, 2X-, or 3XGFP. (A) A 441-nt product (asterisk) establishes the insertion of GFP in germinated spores with 2:2 segregation (see the text). Primers TOR2/+711 and GFP/Rev and spores 2A to 2D (template) were used for colony no. 1 (TB50a/α background). (B) Results of PCR consistent with 2XGFP replacing N321 after the recombination event in JK9 colony no. 1, 1XGFP in TB50 colony no. 2, and 3XGFP in TB50 colony no. 1 (see the text). Primers TOR2/Fwd/+931 and TOR2/Rev/+990 were used. The templates used were as follows: 2XGFP-JK9 lanes, 2A, 2B, none, and 20C; 1XGFP-TB50 lanes, 18A, 18B, 20A, and 20B (colony no. 2); and 3XGFP-TB50 lanes, 2A, 2B, 7C, and 7D (colony no. 1). Arrows indicate diagnostic bands for 1X-, 2X-, and 3XGFP (see the text). (C) Western blot with anti-GFP of 40 μg of lysate protein (see Materials and Methods) from the control (Ctl), TB50a, and VA102 strains.
FIG. 9.
FIG. 9.
TOR2N321-3XGFP localizes to punctate structures near the plasma membrane. We performed microscopy with the following strains in SD or SD-leu (top to bottom): TB50a (control strain, in SD), TOR1-3XGFP (V102, in SD), TOR1-3XGFP plus FYVE-dsRed (VA102 transformed with pTPQ127, in SD-leu), and TOR2-3XGFP plus Sec7-dsRed (VA102 transformed with pTPQ128, in SD-leu). The VA102 strain lost the pSH47 (URA3) plasmid by 5-fluoroorotic acid treatment (Table 3).
FIG. 10.
FIG. 10.
TOR1 and TOR2 have distinct localization patterns by confocal microscopy. The control (TB50), TOR1-3XGFP (VA66), and TOR2-3XGFP strains grown in YPD were imaged by confocal microscopy (see Materials and Methods). The exposure settings used were as follows: Tor1, 400 ms GFP and 50% laser intensity; Tor2, 800 ms GFP and 70% laser intensity; and TB50, 800 ms GFP and 70% laser intensity.
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