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Noda Institute for Scientiﬁc Research GRANT
2011 Research Grant

Study on the yeast signaling pathways responsive to stresses
and starvation
Tatsuya MAEDA
The University of Tokyo, Institute of Molecular and Cellular Biosciences
Research aims
Yeasts are superior host microorganisms suitable for various bioproduction, which have been widely used for fermentation and brew processes. This study addressed stresssensing and regulation mechanisms of two stress-responsive pathways that we have been studying, aiming at the establishment of the theoretical basis to control the cellularresponses to stresses and starvation, which potentially impair productivity in bioproduction processes using yeasts.
For the osmotic stress-responsive HOG pathway, mutants
were isolated and characterized that activate the pathway
even under low osmolarity conditions, to clarify what andhow the sensing machinery monitors as an index of osmoticstress. For the starvation-responsive TOR pathway, novel
regulatory mechanisms were explored by screening for new
regulatory factors. Additionally, the bases for the different
sensitivity to rapamycin, a speciﬁc inhibitor of TOR,
among various cells were also addressed.
Methods
Osmotic stress-responsive HOG pathway
In a host strain for the yeast two-hybrid system expressing a DNA-binding domain-Hog1 fusion protein, the expression of the reporter gene becomes responsive to osmotic stress1). From this reporter strain, mutants were isolated in which the HOG pathway is activated even under
low osmolarity conditions. Then the mutated genes wereidentiﬁed by plasmid complementation. The activation ofthe HOG pathway was monitored by immunoblotting using
an anti-phospho Hog1 antibody. Myriocin and aureobasidin
A were used to inhibit the biosynthesis of sphingolipids.
Starvation stress-responsive TOR pathway
Cells expressing the activated TOR mutant grow poorly
under nutrient-limited conditions due to inappropriately
high activation of the TOR pathway. In order to identify
novel negative regulators of the TOR pathway, genes werescreened for that rescue the poor growth when over-expressed. The localization of the components of the TOR
complex was monitored using functional GFP-fusion constructs. The activation status of the TOR pathway was monitored by immunoblotting using a phospho-speciﬁc antibody against Sch9, a major substrate of TOR. Mutation frequency was measured at the CAN1 locus by monitoring
canavanine resistance.

The growth of ﬁssion yeast was monitored on media containing various concentrations of caffeine and rapamycin incombination. TOR was puriﬁed from ﬁssion yeast cells
using an epitope tag added to TOR, and its in vitro kinase
activity was measured using 4E-BP1 as the substrate. To
examine the rapamycin-sensitivity, rapamycin and GSTFKBP12 were included in the kinase assay.
Results
The osmotic stress-sensing mechanism of the HOG pathway2)
From a reporter strain to monitor the activation of theHOG pathway, mutants were isolated that constitutively activate the pathway even under low osmolarity conditions.
Among the mutants, one that showed clear activation andtemperature sensitive growth was subjected for cloning of
the responsible gene. It turned out to be an lcb2 mutant.
LCB2 encodes a subunit of serine palmitoyltransferase,
which catalyzes the ﬁrst step of the sphingolipid biosynthesis. It was conﬁrmed that cells with the lcb2 mutation constitutively activate Hog1 phosphorylation even under low
osmolarity conditions.
Then, we found that it was decrease in the levels of thecomplex sphingolipids that induces activation of the HOG
pathway, by using inhibitors and mutants of the sphingolipid biosynthesis. Furthermore, inhibition of the ergosterol biosynthesis also induced activation of the pathway.
Sphingolipids and ergosterol are tightly packed together incell membranes, resulting in the formation of membrane
domains called rafts. Sensing components of the HOG
pathway, Sln1 and Sho1, were distributed in detergent-resistant membranes, and therefore appeared to localize torafts. This distribution of Sho1 was enhanced upon not only
osmotic stress but decrease in sphingolipid levels. In contrast, the distribution of Sln1 under the same conditions did
not signiﬁcantly change, but the electrophoretic mobility of
Sln1 on blue native PAGE changed from that of multimer tomonomer.
A novel regulatory mechanism of the TOR pathway mediated by stress granules3)
Stress granules (SGs) are cytoplasmic foci transiently
formed in response to stresses, which contain nontranslating mRNA and translation factors. Overexpression of Pbp1,a component of SG, was found to reduce the activity ofTOR. Then, we found that the TOR complex is sequestered45

into SG, when SG formation is induced by heat stress. Itwas known that the activity of the TOR pathway is transiently inhibited in cells exposed to heat stress. We also
found that the reactivation kinetics of the TOR pathway
during the recovery phase from heat stress was accelerated
when SG formation was inhibited by cycloheximide treatment. This indicates that TOR sequestration into SGs inhibits premature activation of the TOR pathway during therecovery phase. This model was also conﬁrmed using a mutant TOR complex that is reluctant to SG sequestration.
Furthermore, the mechanism contributes to reducing heatinduced mutations.
Bases for cellular rapamycin-sensitivity4)
In many organisms including the yeast Saccharomyces
cerevisiae, cell growth is sensitive to rapamycin treatment.
In contrast, it is not the case in ﬁssion yeast, Schizosaccharomyces pombe, and the bases for this rapamycin-resistancehave attracted wide interests. Recently, it was reported that
rapamycin only partially inhibits the activity of TOR fromthe observations using novel mTOR inhibitors that directly
inhibit mTOR’s kinase activity. It is therefore possible thatﬁssion yeast cells are rapamycin-resistant just because theresidual TOR activity after inhibition by rapamycin isenough to support the growth. To test the possibility, we examined the kinase activity of ﬁssion yeast TOR with an invitro assay and found that it was indeed inhibited by rapamycin of similar concentrations that inhibit TOR in otherorganisms. As the kinase activity of TOR is inhibited alsoby caffeine, caffeine treatment may further reduce the remaining TOR activity after inhibition by rapamycin. The
growth of ﬁssion yeast was indeed inhibited by rapamycinin the presence of a moderate concentration of caffeine,
which by itself did not affect the growth. This inhibition ofgrowth was shown to be mediated by TOR inhibition byusing TOR mutants resistant to each drug. Moreover, starvation responses including G1 arrest, transcription of starvation responsive genes, and autophagy, which are not induced by rapamycin treatment alone, were induced by rapamycin plus caffeine treatment.

Conclusion
The osmotic stress-sensing mechanism of the HOG pathway2)
Sphingolipids regulate the osmosensing machinery of theHOG pathway, probably because the sensing components
localize to rafts and the change in their localization and/orassociation in response to osmotic stress underlies theirstress-sensing mechanism.
A novel regulatory mechanism of the TOR pathway mediated by stress granules3)
The TOR complex is sequestered to stress granules, andwhich determines the reactivation kinetics of the TOR pathway during the recovery phase from the stress. As this regulation in response to heat stress contributes to reducing
heat-induced genomic mutations, its physiological role canbe to balance between repair and growth depending on theseverity of the stress.
Bases for cellular rapamycin-sensitivity4)
The bases for the difference in rapamycin-sensitivity
among different cells have been elusive. We found that it
depends on the different cellular responses to equally reduced TOR activity, but not on the different rapamycin-sensitivity of the TOR complex itself. Moreover, difference inthe rapamycin-sensitivity of various physiological events
controlled by TOR was shown to be just quantitative rather
than qualitative.
References
1)

Alepuz, P.M., de Nadal, E., Zapater, M., Ammerer, G., and Posas, F.
(2003) Osmostress-induced transcription by Hot1 depends on a
Hog1-mediated recruitment of the RNA Pol II. EMBO J. 22: 2433–2442.
2) Tanigawa, M., Kihara, A., Terashima, M., Takahara, T., and Maeda,T. (2012) Sphingolipids regulate the yeast high-osmolarity glycerol
response pathway. Mol. Cell. Biol. 32: 2861–2870.
3) Takahara, T. and Maeda, T. (2012) Transient sequestration ofTORC1 into stress granules during heat stress. Mol. Cell 47: 242–252.
4) Takahara, T. and Maeda, T. (2012) TORC1 of ﬁssion yeast is rapamycin-sensitive. Genes to Cells 17: 698–708.