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Noda Institute for Scientific Research GRANT
2012 Young Investigator Research Grant

Study on the function of sphingolipids during the starvation
response
Keisuke Obara
Faculty of Pharmaceutical Sciences, Hokkaido University

Research aims
Under nutrient-starved conditions, cells draw on a variety of responses to survive. In yeast, starvation causes ashift from fermentation to aerobic respiration. It also causes
large changes in membrane dynamics, as several secreted
proteins are redirected to vacuoles for degradation and autophagy is induced to recycle nutrients. We are currently
studying the role of sphingolipids in the starvation responsein yeast. We previously found that 1) synthesis of a specificsphingolipid species is required for autophagy1) and 2) cells
rapidly die under nitrogen starvation (-N) conditions if
sphingolipid metabolism is perturbed. In this study, we focused on elucidating the importance of sphingolipid metabolism under -N conditions.
Methods
Sphingolipid synthesis (Fig. 1A) was inhibited at each
step, and cell viability was calculated by counting deadcells that were intensely stained with phloxine B. Vacuole
lumens were visualized by using 2′,7′-Dichlorofluoresceindiacetate (DCFDA), which fluoresces green under acidic
conditions.
Results
A mannosylinositol phosphorylceramide synthase mutant
rapidly dies under -N conditions
Mutant cells (csg1∆ csh1∆) that are defective in synthesizing mannosylinositol phosphorylceramide (MIPC) rapidly die under -N conditions (Fig. 1B). Interestingly, csg1∆
csh1∆ cells are resistant to carbon and nitrogen starvation(-NC). This result suggests that some energy-consuming
process takes place during the -N response and that duringthis response, sphingolipid synthesis/metabolism becomes
extremely important for cell survival. We examined the effect of myriocin, an inhibitor of de novo sphingolipid synthesis, on csg1∆ csh1∆ cells under -N conditions, andfound that it greatly reduced cell death. This indicates that
it is the accumulation of the MIPC precursor inositol phosphorylceramide (IPC), rather than the absence of MIPC,that has a role in the rapid cell death that occurs under -N
conditions. Sphingolipids are composed of sphingoid longchain bases (LCBs) and fatty acids, both of which undergohydroxylation. Saccharomyces cerevisiae contains five IPC

species, each differing in the number and/or positions ofhydroxyl groups on the LCB and fatty acid moieties.
MIPC-deficient cells defective in hydroxylation of the LCB
moiety (csg1∆ csh1∆ sur2∆ cells) were resistant to -N (Fig.
1C), indicating that the accumulation of IPCs with hydroxyl groups on the LCB causes cell death under -N conditions.
Intracellular Ca2+ is responsible for MIPC synthase mutant cell death under -N conditions
IPCs with hydroxyl groups on the LCB moiety arethought to cluster through binding to Ca2+ and hydrogenbonding between their hydroxyl groups, leading to local destabilization of membranes.2) Interestingly, csg1∆ csh1∆
cells cultured in medium lacking Ca2+ did not die after
being transferred to -N medium. This result indicates that
intracellular Ca2+ accumulated before undergoing -N is involved in csg1∆ csh1∆ cell death. Since the vacuole is themajor Ca2+ storage organelle in yeast, we visualized thevacuoles of csg1∆ csh1∆ cells under -N conditions. In approximately half of the dying cells, vacuolar contents
leaked prior to the death and shrinkage of the cells. Thus, atleast in half of the dead cells, the leakage of vacuolar contents seems to be the trigger for cell death. Topologically,
IPC is localized to the luminal (extracytosolic) leaflet of thelipid bilayers of the vacuolar membrane. Therefore, Ca2+
might directly bind to IPC in the luminal leaflet, leading to
the destabilization of the vacuolar membrane. Secretory
vesicles are redirected to the vacuole under -N conditions.3)
We speculate that, because of this rearrangement in membrane dynamics, IPC over-accumulates at the vacuolar
membrane in csg1∆ csh1∆ cells under -N conditions. Theaccumulated IPC may then cluster excessively throughbinding to Ca2+ and hydrogen binding between the hydroxyl
groups, and this clustering culminates in a local disruptionof the vacuolar membrane (Fig. 2).2)
The inactivation of TOR complex 2 leads to MIPC synthase mutant cell death
TOR complexes (TORC1 and TORC2) regulate various
processes including protein synthesis and nutrient starvation. Therefore, we examined whether the rapid death ofcsg1∆ csh1∆ cells under nutrient starvation occurs throughthe inactivation of TOR complexes. Specific inactivation ofTORC2 but not TORC1 caused the rapid death of csg1∆
csh1∆ cells, even in nutrient-rich medium. This result

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Fig. 1. Accumulation of IPC leads to cell death through leakage of vacuolar contents. A. Sphingolipid synthesis in yeast. B. Cells at log phase were transferred to -N medium and cultured for 4 h. Dead cells were stained with phloxine B. Bar, 5 μm. C. MIPC-deficient cells defective in hydroxylation of LCB(csg1∆ csh1∆ sur2∆), as well as wild-type (WT) and csg1∆ csh1∆ cells, were cultured in -N medium, and their viability was calculated at the indicated time.
D. Vacuoles of csg1∆ csh1∆ mutant cells (v) were visualized with DCFDA and observed successively at 2 h of -N. In approximately half of the dying cells,
vacuolar contents leaked (arrowhead) prior to the death and shrinkage of the cell. The time when the leakage of vacuolar contents became obvious was defined as 0 min.

Fig. 2. Working hypothesis. -N invokes certain cellular responses, some of which are induced through the inactivation of TOR complexes. In the earlystarvation responses that are downstream of TORC2 inactivation, the conversion of IPC to MIPC is essential for cell survival. Perturbation of IPC–MIPC
conversion results in accumulation in the vacuolar membrane of IPC species that have hydroxyl groups on their LCB moieties. This causes the disruption ofthe vacuolar membrane through excessive clustering of IPC via binding to Ca2+ and hydrogen bonding between hydroxyl groups. On the other hand, de novo
synthesis of IPC is required for autophagy,1) which is induced through TORC1 inactivation. In summary, the appropriate flow of sphingolipid synthesis andmetabolism is extremely important for starvation responses occurring through TOR complexes.

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strongly suggests that the rapid death of csg1∆ csh1∆ cells
under -N conditions is a downstream event of TORC2 inactivation.

cumulation of IPC at the vacuolar membrane may have afatal effect, i.e., the leakage of vacuolar contents to thewhole cell through some mechanism involving Ca2+.

Conclusion

References

Under -N conditions, the conversion of IPC to MIPC is a
critical step for cell survival.2) On the other hand, de novo
synthesis of IPC is required for autophagy.1) Thus, the synthesis and metabolism of sphingolipids is extremely important for early starvation responses. Processes that require
normal IPC–MIPC conversion are likely downstream ofTORC2 inactivation. During such processes, excessive ac-1) Yamagata, M., Obara, K., and Kihara, A. (2011) Sphingolipid synthesis is involved in autophagy in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 410: 786–791.
2) Yamagata, M., Obara, K., and Kihara, A. (2013) Unperverted synthesis of complex sphingolipids is essential for cell survival under nitrogen starvation. Genes Cells 18: 650–659.
3) Hamasaki, M., Noda, T., Baba, M., and Ohsumi, Y. (2005) Starvation triggers the delivery of the endoplasmic reticulum to the vacuole
via autophagy in yeast. Traffic 6: 56–65.