Role of the TOR signaling pathway in the freeze-thaw stressresponse in yeast
Yoshiharu INOUE
Graduate School of Agriculture, Kyoto University
Research aims
Living organisms survive by adapting to changes in the environment. Changes inthe temperature of the growth environment and changes in nutritional status are sensed by
the corresponding sensors, and gene expression and metabolic regulation suitable for such anenvironment are performed. For example, the expression of heat shock proteins increases
when the temperature of the growth environment is elevated, and the mechanism is wellstudied from E. coli to human. However, freeze-thaw stress is one of the most frequently
encountered environmental stresses for cells in both natural and laboratory environments.
Yeast encounter freeze-thaw stress in the industrial field also, such as in frozen dough.
Nonetheless, studies on the freeze-thaw stress response are currently delayed compared tothat on heat (high temperature) shock stress.
Yeast cells in the stationary phase of growth show higher freeze-thaw stress
tolerance than cells in the logarithmic growth phase. Usually, yeast cells in the logarithmic
growth phase contain 2 to 3 fragmented vacuoles. In contrast, in stationary phase cells, themorphology of the vacuole changes into one enlarged shape. In this study, we focused on therelationship between vacuolar morphology and freeze-thaw stress tolerance. However,another difference in the environment of yeast culture between the logarithmic growth phaseand the stationary phase is the availability of nutrients. The TOR (target of rapamycin)
pathway plays a pivotal role in transmitting nutrient signals in eukaryotes. The TOR signal isactivated in the presence of sufficient nutrients but is inactivated when nutrients are depleted.
The second purpose of this study was to explore the relationship between the TOR signaling
pathway and the freeze-thaw stress response.
Methods
A freezing tolerance test was carried out as follows: yeast cells were cultured in
YPD medium (2% glucose, 1% yeast extract, 2% peptone; pH 5.5) to log phase. After
collecting cells by centrifugation, cells were washed with sterilized 50 mM potassium
phosphate buffer (pH 6.5), quickly frozen at -30 ºC, and stored for 1-3 days. Cells werethawed at 25 ºC for 20 min in a heat block, diluted with sterilized 0.85% NaCl solution, andthen spread on YPD agar plates. Cells were cultured at 28 ºC for several days to form

colonies, and the survival rate was compared with the survival rate of the samples before
freezing.
Results
1. Vps34 and freeze-thaw stress resistance
One of the morphological features of yeast cells in the stationary phase of growthis the enlargement of vacuoles. Among the mutants defective in vacuolar shape, the mutants
classified into class D (vps8∆, vps9∆, vps15∆, vps34∆, vps45∆, fab1∆, and vac14∆) thathave an enlarged vacuole even in the logarithmic growth phase, were subjected to thefreezing tolerance test. As a result, cells carrying enlarged vacuoles did not necessarily
acquire tolerance to freeze-thaw stress, but the mutants defective in phosphatidylinositol
3-kinase (Vps34) and its regulator protein (Vps15) showed significantly higher freeze-thaw
stress tolerance, even in the logarithmic growth phase.
Next, to examine whether the lipid kinase activity of Vps34 was necessary for
acquiring freeze-thaw stress tolerance, the freezing tolerance test was done using aVps34N736K mutant defective in lipid kinase activity1). The vps34∆ mutant carrying the vector
showed high freeze-thaw stress tolerance, whereas the mutant with the wild-type VPS34
gene lost freezing tolerance. The Vps34N736K mutant exhibited high freeze-thaw stresstolerance. Phosphatidylinositol-3-phosphate (PI3P) produced by the Vps34 reaction isconverted to phosphatidylinositol-3,5-bisphosphate by the action of Fab1. Since theFab1-deficient mutant did not exhibit freeze-thaw stress tolerance, PI3P seemed to beinvolved in the acquisition of freeze-thaw stress resistance.
2. TOR signaling and freeze-thaw stress resistance
One of the environmental changes in stationary phase yeast culture is a decrease(depletion) of nutrients inside and outside the cell. Thus, the freezing tolerance wascompared after transferring logarithmic growth phase cells cultured in nutrient medium tomedium lacking carbon or nitrogen sources. As a result, freeze-thaw stress tolerance wasacquired when cells were transferred to the medium depleted of nutrients.
TOR signaling is a nutrient signaling pathway evolutionally conserved ineukaryotes. TOR is a Ser/Thr kinase and forms two functionally distinct TOR complexes(TORC1 and TORC2). Of these, the TOR complex that transmits nutrient signals is TORC1.
TORC1 is activated under conditions where nutrients are sufficiently present but isinactivated when nutrients are depleted. Rapamycin is a specific inhibitor of TORC1. To
explore the correlation between TORC1 inhibition and freeze-thaw stress tolerance,
rapamycin was added to yeast culture medium in which a sufficient amount of nutrients waspresent, and then the freezing tolerance test was conducted. As a result, rapamycin-treatedcells acquired freeze-thaw stress tolerance. Taken together, these results suggest that that theinhibition of the TOR signaling pathway is involved in the acquisition of freezing tolerance.
3. Functional correlation between Vps34 and TORC1
Since both Vps34-deficiency and TORC1-dysfunction gave a common phenotypein terms of freezing tolerance, the possibility of a functional correlation between Vps34 andTORC1 was suspected. To investigate the effect of Vps34-deficiency on the kinase activityof TORC1, the phosphorylation state of Sch9, which is a substrate of TORC1, was examined.
Sch9 was in a dephosphorylated state in the Vps34-deficient mutant, suggesting that Vps34
was involved in the activation of the TOR kinase.
TORC1 activation is regulated by two distinct pathways, i.e. a small G protein
Gtr1-dependent manner and a vacuolar membrane-associated protein Pib2-dependent manner.
Pib2 contains a PI3P-binding domain (FYVE domain)2). We examined the effect of deletionof Gtr1 and Pib2 on freeze-thaw stress resistance. A pib2∆ mutant did not show freezing
tolerance, but a gtr1∆ mutant did; however, the extent of the freeze-thaw stress tolerance ofthe gtr1Δ mutant was not as high as that of the vps34Δ mutant. These results suggested thatthe TORC1 signaling pathway involved in freeze-thaw stress tolerance was dependent on
Vps34 but was different from the Pib2 pathway.
Conclusion
Vps34 regulated TORC1 activity via PI3P, and TORC1 negatively regulated thefreeze-thaw stress response in yeast. Thus, it seems that the TORC1 regulatory mechanism
involved in the freeze-thaw stress response was regulated in a Pib2-independent manner.
References
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