LABORATORY OF BIOLOGICAL DIVERSITY
KAMADA Group
Assistant Professor:

KAMADA, Yoshiaki

Tor (target of rapamycin) is a Ser/Thr protein kinase
which is well conserved in organisms spanning from yeasts
to mammals. Tor forms two distinct protein complexes,
TORC1 (Tor complex1) and TORC2. On one hand, TORC1
is involved in amino acid sensing, regulation of protein
synthesis (especially the translation step), the cell cycle, andautophagy. On the other hand, TORC2 is responsible foractin organization and cell integrity. Up until now, it’s been
deemed unlikely that TORC2 can recognize nutrient signals.
The aim of our research group is to reveal the molecularmechanisms underlying how TORC1 receives amino acidsignals and how the TORC1/2 pathways regulate phenomena
they are associated with. We have been studying Tor signaling in the budding yeast Saccharomyces cerevisiae, and havefound three novel TOR signaling pathway branches (Figure 1).

product of ARS acts as an amino acid signal rather than anamino acid itself. Biochemical in vitro TORC1 assay also
revealed that tRNA directly inhibits TORC1 kinase activity,
suggesting that uncharged tRNA, produced under aminoacid-depleted condition, functions as a starvation signal.
Reducing cellular tRNA molecules desensitizes TORC1 inactivation by nitrogen starvation in vivo.
Based on these results, a TORC1 regulatory model wasproposed which contends that free tRNA released fromprotein synthesis under amino acid starvation inhibits
TORC1 activity. Therefore, TORC1 employs a tRNAmediated mechanism to monitor intracellular amino acids(Figure 2).

Figure 2. A schematic model of how amino acid is sensed by TORC1.
Cytosolic free tRNA inactivates TORC1 under amino acid limited condition.

Since tRNA directly inhibits TORC1 activity, TORC1
should have a tRNA-binding site(s). Thus, we have investigated further so as to determine the tRNA-binding site. Sofar, we have obtained a good candidate for the tRNA-bindingsite in Tor protein, and we will now focus on this domain todetermine its function.

II. TORC1 phosphorylates Atg13, the molecularswitch of autophagy

Figure 1. Tor signaling pathway for budding yeast. Our group has foundthree branches of the Tor pathway.

I. How do amino acids regulate TORC1?

TORC1 is regulated by amino acids which in themselves
are fundamental nutrients. 20 species of amino acids thatbuild proteins cannot be interchanged with each other.
Therefore, each amino acid must be individually detected byTORC1.
We have conducted genetic research and discovered theinvolvement of (aminoacyl-) tRNA in TORC1 regulation.
For example, mutants of aminoacyl-tRNA synthetases(ARSs) exhibited inactivation of TORC1 even under aminoacid-rich condition, suggesting that aminoacyl-tRNA, aTORC1 negatively regulates autophagy: a protein degradation system induced by nutrient starvation.
We have been consequently able to discover the TORC1mediated regulatory mechanism of autophagy. Under nutrient-rich conditions, TORC1 directly phosphorylates Atg13,
a component of the Atg1 kinase complex. Atg1 is a Ser/Thr protein kinase, the activity of which is essential forautophagy and is largely enhanced in response to TORC1
inactivation. Activation of Atg1 requires formation of theAtg1 complex. Phosphorylation of Atg13 by TORC1 playsa pivotal role in Atg1 complex formation; phosphorylated
Atg13 loses its affinity to Atg1 resulting in disassembly of
the Atg1 complex and repression of autophagy. On the otherhand, dephosphorylation of Atg13 triggers formation of theAtg1 complex, activation of Atg1 kinase, and consequent
induction of autophagy.

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National Institute for Basic Biology

Evolutionary Biology and Biodiversity

III. Relationships between TORC1 and eIF4Ebinding proteins

EIF4E-binding proteins (4EBPs), which binds to translation initiation factor 4E (eIF4E), are thought to negatively
regulate translation initiation, because their competition
infused binding to eIF4E prevents eIF4E-eIF4G association,
which is a primary and essential procedure in translation
initiation. In mammals, the inhibitory function of 4EBP1
is regulated by mammalian TORC1 (mTORC1). Under
nutrient-rich conditions, mTORC1 phosphorylates 4EBP1,
and the phosphorylated 4EBP1, lose affinity to eIF4E and let
eIF4E bind to eIF4G.
We examined whether two yeast 4EBPs, Caf20 and Eap1,
have properties in common with 4EBP1. Caf20, but not
Eap1, is phosphorylated in a TORC1-dependent manner, itbinds to eIF4E, and it never associates to eIF4G. However,Caf20 was not directly phosphorylated by TORC1, Caf20eIF4E binding was not affected by TORC1 activity, andCaf20-eIF4E complex was found in ribosome fraction, suggesting that the function of the yeast 4EBP is different fromthat of its mammalian counterparts.

Publication List:〔Original paper〕

T., Ishii, T., Kamada, Y., Funakoshi, M., Kobayashi, H.,• Sekiguchi,and Furuno, N. (2022). Involvement of Gtr1p in the oxidative stressresponse in yeast Saccharomyces cerevisiae. Biochem. Biophys. Res.
Commun. 598, 107–112. DOI: 10.1016/j.bbrc.2022.02.016

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