Functional analysis of TOR complex 2 and its control of sphingolipid biosynthesis in "Saccharomyces cerevisiae"

sphingoid base, one of the intermediate of sphingolipids metabolites, plays a key role as a signaling molecule which activate PKH kinases and its substrates YPKs and PKC1 (Casamayor, Torrance et al. 1999; Friant, Lombardi et al. 2001; Schmelzle, Helliwell et al. 2002). Endocytosis The initial observation that sphingolipids mediate endocytosis came up with a genetic screening for endocytosis mutants (Munn and Riezman 1994); one of those mutants was allelic to the LCB1 (Sutterlin, Doering et al. 1997). Friant et al. 2000). The endocytic defect of the lcb1 mutant, named lcb1-100, could be overcome by exogenous sphingoid base (Zanolari, Even when endogenous sphingolipid synthesis was inhibited, and These results exogenous sphingolipid could not be given a further modification, sphingoid bases were able to suppress the endocytic defect (Zanolari, Friant et al. 2000). suggest that sphingoid base is required for the internalization step of endocytosis. Consistent with the study that showed that sphingoid base can activate PKH1 and PKH2, overexpression of either PKH1 or PKH2 can suppress the lcb1-100 endocytic defect (both fluid phase and receptor mediated internalization) and temperature sensitivity (Friant, Lombardi et al. 2001; deHart, Schnell et al. 2002). In addition, a temperature sensitive mutant pkh-ts, harboring a chromosomal deletion of PKH2 (pkh2::LEU2) and a temperature-sensitive pkh1-ts mutant allele, pkh1D398G (Inagaki, Schmelzle et al. 1999), failed in fluid phase and receptor mediated internalization at non-permissive temperature 37°C (Friant, Lombardi et al. 2001). Not only PKH kinases, but also YPK1 and YPK2 were found to be required for endocytosis (deHart, Schnell et al. 2002). These findings suggest that sphingoid bases mediate endocytosis via the protein kinases PKH1 and PKH2. Since endocytosis is tightly coupled with actin assembly (Engqvist-Goldstein and Drubin 2003), it is conceivable that sphingolipids mediate actin cytoskeleton organization. Indeed, the serine palmitoyltransferase temperature sensitive mutant lcb1-100 showed actin depolarization at non-permissive temperature. This actin defect was suppressed by additional sphingoid bases (Zanolari, Friant et al. 2000) and by overexpression of either PKH1, PKH2, YCK2 or PKC1 (Friant, Zanolari et al. 2000; Friant, Lombardi et al. 2001). Thus, sphingoid bases and PKH-PKC signaling cascade biosynthesis. Our findings in this part of the thesis indicate that TORC2, but not
TORC1, mediate sphingolipid biosynthesis.