Amino acid residues required for Gtr1p-Gtr2p complex formation and its interactions with the Ego1p-Ego3p complex and TORC1 components in yeast.
The yeast Ras-like GTPases Gtr1p and Gtr2p form a heterodimer, are implicated in the regulation of TOR complex 1 (TORC1) and play pivotal roles in cell growth. Gtr1p and Gtr2p bind Ego1p and Ego3p, which are tethered to the endosomal and vacuolar membranes where TORC1 functions are regulated through a relay of amino acid signaling interactions. The mechanisms by which Gtr1p and Gtr2p activate TORC1 remain obscure. We probed the interactions of the Gtr1p-Gtr2p complex with the Ego1p-Ego3p complex and TORC1 subunits. Mutations in the region (179-220 a.a.) following the nucleotide-binding region of Gtr1p and Gtr2p abrogated their mutual interaction and resulted in a loss in function, suggesting that complex formation between Gtr1p and Gtr2p was indispensable for TORC1 function. A modified yeast two-hybrid assay showed that Gtr1p-Gtr2p complex formation is important for its interaction with the Ego1p-Ego3p complex. GTP-bound Gtr1p interacted with the region containing the HEAT repeats of Kog1p and the C-terminal region of Tco89p. The GTP-bound Gtr2p suppressed a Kog1p mutation. Our findings indicate that the interactions of the Gtr1p-Gtr2p complex with the Ego1p-Ego3p complex and TORC1 components Kog1p and Tco89p play a role in TORC1 function.
Department of Molecular Biology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.
Takeshi
T
Sekiguchi
Yoshiaki
Y
Kamada
Nobuaki
N
Furuno
Minoru
M
Funakoshi
Hideki
H
Kobayashi
D000596:Amino Acids / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D055503:Protein Multimerization; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
D000596:Amino Acids; C121263:GTR2 protein, S cerevisiae; C552591:Gse1 protein, S cerevisiae; C121262:Gtr1 protein, S cerevisiae; C501979:MEH1 protein, S cerevisiae; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; C561842:TORC1 protein complex, S cerevisiae; D014157:Transcription Factors; D020559:Monomeric GTP-Binding Proteins