A New Crosslinking Assay to Study Guanine Nucleotide Binding in the Gtr Heterodimer of S. cerevisiae

ABSTRACT

The mechanistic target of rapamycin (mTOR) complex is responsible for coordinating nutrient availability with eukaryotic cell growth. Amino acid signals are transmitted towards mTOR via the Rag/Gtr heterodimers. Due to the obligatory heterodimeric architecture of the Rag/Gtr GTPases, investigating their biochemical properties has been challenging. Here, we describe an updated assay that allows us to probe the guanine nucleotide-binding affinity and kinetics to the Gtr heterodimers in Saccharomyces cerevisiae. We first identified the structural element that Gtr2p lacks to enable crosslinking. By using a sequence conservation-based mutation, we restored the crosslinking between Gtr2p and the bound nucleotides. Using this construct, we determined the nucleotide-binding affinities of the Gtr heterodimer, and found that it operates under a different form of intersubunit communication than human Rag GTPases. Our study defines the evolutionary divergence of the Gtr/Rag-mTOR axis of nutrient sensing.

KEYWORDS:

• GTPase
• Gtr GTPase
• Rag GTPase
• enzyme kinetics
• guanine nucleotide
• crosslinking
• mTOR
• nutrient sensing

Acknowledgments

We thank all members of the Shen Laboratory for their helpful insights and I. Garcia for critical reading of the manuscript. This work was supported by grants from the American Heart Association (928304) and the National Institute of General Medical Sciences of the National Institutes of Health (R35 GM146824) to K.S.

Author contributions

D.D.D. and K.S. conceptualized the project. D.D.D. and K.V. performed the experiments and analysed the data. D.D.D. and K.S. wrote and edited the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the American Heart Association [928304]; National Institute of General Medical Sciences [R35 GM146824].