Abstract

Target of rapamycin complex 1 (TORC1) is a conserved protein kinase complex whose activity controls metabolism in eukaryotic cells. To function, TORC1 and its regulators are localized to lysosome membranes. The mechanism(s) that control TOR complex protein lifetimes remain enigmatic. The IntraLumenal Fragment (ILF) pathway is a selective protein degradation pathway that involves lysosomes. Thus, I hypothesized that components of TORC1, EGO and SEA complexes may be degraded by this ILF pathway.
Using the yeast S. cerevisiae and its vacuolar lysosome (or vacuole) as models, I tested this hypothesis by first imaging live cells by fluorescence microscopy. I found that several subunits of TORC1, EGO and SEA complexes tagged with GFP are sorted into boundaries between docked vacuoles and accumulate within the vacuole lumen. However, when I isolated vacuoles from cells and repeated this experiment in vitro, I found that nearly all components re-localized to endosomes. The only exception was Ego3, a subunit that tethers the EGO complex to vacuole membranes. I then applied rapamycin, an inhibitor of TOR, to induce TOR protein downregulation and degradation. In vivo, I found that rapamycin stimulated sorting of TOR complex . Whereas, in vitro, rapamycin only enhanced sorting, internalization and degradation of Ego3.
In conclusion, many components of TOR signaling complexes seem to be degraded by the ILF pathway including Ego3. Thus, I speculate that the ILF pathway may play an important role in downregulating TOR activity.