Characterization of the yeast rapamycin-sensitive phosphoproteome

Cell growth is a tightly regulated process, where a cell adapts its growth
according to nutrient availability and cellular stress. Tor (Target Of Rapamycin) is an
evolutionary conserved protein kinase and central controller of cell growth. It is found in
two functionally distinct protein complexes termed TORC1 and TORC2. Rapamycinsensitive
TORC1 mediates temporal control of cell growth whereas rapamycininsensitive
TORC2 mediates spatial control of cell growth. Although many cellular
processes regulated by TORC1 have been identified, the molecular mechanisms by which
TORC1 signals to these diverse processes are not well understood. For example, only few
substrates of either TORC1 or its direct effector SCH9 are known in the yeast S.
cerevisiae. To identify novel TORC1 targets in a global manner, a quantitative
phosphoproteomic strategy was established, which allowed to reproducibly relative
quantify more than 2,500 phosphorylation sites in untreated and rapamycin-treated cells.
In parallel, a proteomic study was performed to monitor changes in protein abundances
induced by rapamycin treatment. In total 55 and 78 proteins were significantly less
respectively more phosphorylated upon rapamycin treatment in the phosphoproteomic
analysis. Among them there were many proteins already linked to the TORC1 signaling
pathway, which functioned as internal control. Many regulated proteins were
transcription factors or kinases, which are often present at low copy number in the cell,
suggesting an in-depth analysis of the yeast phosphoproteome. Among the
hypophosphorylated phosphopeptides the PKA consensus motif was significantly overrepresented
suggesting a cross-talk between the TORC1 and PKA signaling pathways.
This hypothesis was further supported at the molecular level for the protein Maf1, Ksp1
and Ypk3. In addition, to validate and better characterize the phosphoproteomic data,
more targeted experiments for some of the regulated phosphoproteins were performed.
This revealed the involvement of novel proteins (like Hal5, Isw2, Kkq8, Ldb19, Mtc1,
Noc2 and Vtc2) in TORC1 signaling. On these proteins, several rapamycin-regulated
phosphorylation sites were mapped and their absolute phosphorylation occupancy was
estimated. Interestingly, rapamycin-regulated phosphorylation sites usually exhibited low to moderate stoichiometries. Subsequent mutagenesis experiment will address the
involvement of those specific phosphorylation sites in TORC1 signaling.