Analysis of TORC1-body Formation in Budding Yeast.

The Target of Rapamycin kinase Complex I (TORC1) is the master regulator of cell growth and metabolism in eukaryotes. In the presence of pro-growth hormones and abundant nutrients, TORC1 is active and drives protein, lipid, and nucleotide synthesis by phosphorylating a wide range of proteins. In contrast, when nitrogen and/or glucose levels fall, TORC1 is inhibited, causing the cell to switch from anabolic to catabolic metabolism, and eventually enter a quiescent state. In the budding yeast Saccharomyces cerevisiae, TORC1 inhibition triggers the movement of TORC1 from its position around the vacuole to a single focus/body on the edge of the vacuolar membrane. This relocalization depends on the activity of numerous key TORC1 regulators and thus analysis of TORC1 localization can be used to follow signaling through the TORC1 pathway. Here we provide a detailed protocol for measuring TORC1 (specifically, Kog1-YFP) relocalization/signaling using fluorescence microscopy. Emphasis is placed on procedures that ensure: (1) TORC1-bodies are identified (and counted) correctly despite their relatively low fluorescence and the accumulation of autofluorescent foci during glucose and nitrogen starvation; (2) Cells are kept in log-phase growth at the start of each experiment so that the dynamics of TORC1-body formation are monitored correctly; (3) The appropriate fluorescent tags are used to avoid examining mislocalized TORC1.