Figure 1.
Functional GFP-tagged TORC1 and TORC2 subunits localize to the vacuolar and the plasma membrane, respectively. (a) GFP-tagged TORC2 subunits Avo1, Avo2, Avo3, and Bit61 support normal yeast growth, whereas LST8-GFP and KOG1-GFP are temperature-sensitive alleles. The indicated subunits of TOR complexes were tagged with GFP in their endogenous locus (see Materials and Methods). The resulting strains were grown in Log-phase, serially diluted, spotted on YPD medium and grown at 30°C (left) and under heat shock conditions at 37°C (right). (b) Kog1-GFP, Lst8-GFP, and Bit61-GFP localize to distinct places. Cells expressing the indicated GFP fusions were analyzed by confocal microscopy. Representative fluorescent images (bottom) and transmitted light images (top) are shown. Bar, 5 μm. (c) Kog1-GFP localizes to the vacuolar membrane. Kog1-GFP (left) localization was analyzed by fluorescence microscopy in cells with FM4-64 marked vacuoles (middle). The right panel shows an overlay of the channels. Representative confocal midsections are shown. Insets indicate higher magnification of the indicated part of the image. Bar, 5 μm. (d) Bit61-GFP localizes to the plasma membrane. Bit61-GFP (left) localization was analyzed by fluorescence microscopy in cells expressing Pma1-RFPmars (middle). The right panel shows an overlay of the channels. Representative confocal midsections are shown. Insets indicate higher magnification of the indicated part of the image. Bar, 5 μm.