Tor, a Phosphatidylinositol Kinase Homologue, Controls Autophagy in Yeast

Takeshi Noda,Yoshinori Ohsumi

doi:10.1074/jbc.273.7.3963

Takeshi Noda, Yoshinori Ohsumi

Open Access

https://doi.org/10.1074/jbc.273.7.3963

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Abstract

Autophagy is a bulk protein degradation process that is induced by starvation. The control mechanism for induction of autophagy is not well understood. We found that Tor, a phosphatidylinositol kinase homologue, is involved in the control of autophagy in the yeast, Saccharomyces cerevisiae. When rapamycin, an inhibitor of Tor function, is added, autophagy is induced even in cells growing in nutrient-rich medium. A temperature-sensitive tor mutant also leads to induction of autophagy at a nonpermissive temperature. These results indicate that Tor negatively regulates the induction of autophagy. Tor is the first molecule that is identified as a pivotal player in the starvation-signaling pathway of autophagy. Furthermore, we found that a high concentration of cAMP is inhibitory for induction of autophagy. APG gene products are involved in autophagy induced by starvation. Autophagy was not induced in apg mutants in the presence of rapamycin, indicating that the site of action of Tor is upstream of those of Apg proteins. In nutrient-rich medium, Apg proteins are involved also in the transport of aminopeptidase I from the cytosol to the vacuole. Tor may act to switch Apg function between autophagy and transport of aminopeptidase I.

Highlights

When nutrients are depleted from the environment, mammalian as well as yeast cells begin to degrade their own cytosol and organelles
Autophagy in yeast is induced under starvation conditions [10] and can be followed as an accumulation of autophagic bodies in the vacuole when vacuolar degradation is inhibited by inactivation of proteinase B (Fig. 1A)
When rapamycin was added to cells growing in YPD, it was found that autophagic bodies accumulate in the vacuole, indicating that autophagy was induced (Fig. 1C)

Summary

EXPERIMENTAL PROCEDURES

TN127 cells were constructed by disrupting the FKB1 gene of TN125 cells using the plasmid fkb1URA3, a gift from Dr Tanida (Tonen Corporation). TN128 cells were made by replacing the PHO8 gene with pho8⌬60 as above in SH221 (leu 112 ura rme trp his HMLa ade2⌬ tor1::HIS3–3 tor2::ADE2–3/YCplac111::tor2–21ts), a gift from Dr M. The cells in log phase were washed with water and suspended in SD(ϪN) medium (0.17% yeast nitrogen base without amino acids and ammonium sulfate (Difco) and 2% glucose). Rapamycin (Sigma) was added at a final concentration of 0.2 ␮g/ml using a stock solution of 20 ␮g/ml in 90% ethanol and 10% Triton X-100. Flow Cytometry—Cultures (0.3 ml) of yeast in YPD medium (A600 ϭ 0.5) were added to 0.7 ml of ethanol.

Tor Control of Autophagy in Yeast

RESULTS

DISCUSSION