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.