An important signaling pathway involved in the regulation of autophagy is the Ras/PKA pathway (Budovskaya et al., 2004). Inactivation of the Ras/PKA pathway, by overexpression of a dominant-negative allele of RAS2, known as RAS2ala22, resulted in increased induction of autophagy as compared with WT. However, additional inactivation of the genes encoding the PKA catalytic subunits, TPK1, TPK2 and TPK3, in the double Δipt1Δskn1 deletion mutant did not result in an enhanced autophagy phenotype (data not shown) as compared with the double Δipt1Δskn1 deletion mutant, indicating that Skn1, together with Ipt1, might act in the same pathway as Ras/PKA regarding induction/regulation

of autophagy. Moreover, PKA and Sch9 signaling pathways are known to regulate autophagy cooperatively in yeast (Yorimitsu et al., 2007). Long-chain bases including phytosphingosine Histone Methyltransferase inhibitor are recognized as regulators of AGC-type protein

selleck chemical kinase (where AGC stands for protein kinases A, G and C) Pkh1 and Pkh2, which are homologues of mammalian phosphoinositide-dependent protein kinase 1 (Sun et al., 2000). Based on in vitro data, Liu et al. (2005a, b) demonstrated that phytosphingosine stimulates Pkh1 to activate additional downstream kinases including Ypk1, Ypk2 and Sch9, and additionally, that phytosphingosine can directly activate Ypk1, Ypk2 and Sch9. In conclusion, it could be that the higher basal levels of phytosphingosine, which we observed in the double Δipt1Δskn1 mutant, affect Sch9 function directly or (-)-p-Bromotetramisole Oxalate indirectly,

and concomitantly, the authophagy response. Hence, future research will be directed towards determining whether Sch9 or other kinases are part of the link between sphingolipids and autophagy in yeast. In conclusion, all the data obtained in this study point to a negative regulation of autophagy by both Ipt1 and Skn1 in yeast, which could be mediated by sphingoid bases and might act in the same pathway as the Ras/PKA signaling pathway. Apparently, Ipt1 and Skn1 can functionally complement each other under nutrient limitation, not only regarding synthesis of the complex sphingolipid M(IP)2C upon nutrient limitation in half-strength PDB (Thevissen et al., 2005) but also regarding the negative regulation of autophagy under N starvation, as demonstrated in this study. This work was supported by a grant from FWO-Vlaanderen (research project G.0440.07) to B.P.A.C. Postdoctoral fellowships to A.M.A. (Research Council) and to K.T. (Industrial Research Found), both from K.U. Leuven, are gratefully acknowledged. F.M. and D.C.-G. are grateful to the FWF for SFB ‘Lipotox’ and NRN S-9304-B05. Lipidomics CORE at the Medical University of South Carolina is supported by NIH Grant No. C06 RR018823. D.J.K. is supported by National Institutes of Health Public Health Service grant GM53396.