Structure of the Schizosaccharomyces pombe Gtr-Lam complex reveals evolutionary divergence of mTORC1-dependent amino acid sensing.

mTORC1 is a protein kinase complex that controls cellular growth in response to nutrient availability. Amino acid signals are transmitted toward mTORC1 via the Rag/Gtr GTPases and their upstream regulators. An important regulator is LAMTOR, which localizes Rag/Gtr on the lysosomal/vacuole membrane. In human cells, LAMTOR consists of five subunits, but in yeast, only three or four. Currently, it is not known how variation of the subunit stoichiometry may affect its structural organization and biochemical properties. Here, we report a 3.1 Å-resolution structural model of the Gtr-Lam complex in Schizosaccharomyces pombe. We found that SpGtr shares conserved architecture as HsRag, but the intersubunit communication that coordinates nucleotide loading on the two subunits differs. In contrast, SpLam contains distinctive structural features, but its GTP-specific GEF activity toward SpGtr is evolutionarily conserved. Our results revealed unique evolutionary paths of the protein components of the mTORC1 pathway.

Redundant electrostatic interactions between GATOR1 and the Rag GTPase heterodimer drive efficient amino acid sensing in human cells.

Cells need to coordinate nutrient availability with their growth and proliferation. In eukaryotic cells, this coordination is mediated by the mechanistic target of the rapamycin complex 1 (mTORC1) pathway. mTORC1 activation is regulated by two GTPase units, the Rag GTPase heterodimer and the Rheb GTPase. The RagA-RagC heterodimer controls the subcellular localization of mTORC1, and its nucleotide loading states are strictly controlled by upstream regulators including amino acid sensors. A critical negative regulator of the Rag GTPase heterodimer is GATOR1. In the absence of amino acids, GATOR1 stimulates GTP hydrolysis by the RagA subunit to turn off mTORC1 signaling. Despite the enzymatic specificity of GATOR1 to RagA, a recent cryo-EM structural model of the human GATOR1-Rag-Ragulator complex reveals an unexpected interface between Depdc5, a subunit of GATOR1, and RagC. Currently, there is no functional characterization of this interface, nor do we know its biological relevance. Here, combining structure-function analysis, enzymatic kinetic measurements, and cell-based signaling assays, we identified a critical electrostatic interaction between Depdc5 and RagC. This interaction is mediated by the positively charged Arg-1407 residue on Depdc5 and a patch of negatively charged residues on the lateral side of RagC. Abrogating this interaction impairs the GAP activity of GATOR1 and cellular response to amino acid withdrawal. Our results reveal how GATOR1 coordinates the nucleotide loading states of the Rag GTPase heterodimer, and thus precisely controls cellular behavior in the absence of amino acids.

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

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.

Purification and biochemical characterization of the Rag GTPase heterodimer.

The mechanistic target of rapamycin complex 1 (mTORC1) senses nutrient levels in the cell and based on the availability, regulates cellular growth and proliferation. Its activity is tightly modulated by two GTPase units, the Rag GTPases and the Rheb GTPase. The Rag GTPases are the central hub of amino acid sensing as they summarize the amino acid signals from upstream regulators and control the subcellular localization of mTORC1. Unique from canonical signaling GTPases, the Rag GTPases are obligatory heterodimers, and the two subunits coordinate their nucleotide loading states to regulate their functional states. Robust biochemical analysis is indispensable to understanding the molecular mechanism governing the GTPase cycle. This chapter discusses protocols for purifying and biochemically characterizing the Rag GTPase heterodimer. We described two purification protocols to recombinantly produce the Rag GTPase heterodimer in large quantities. We then described assays to quantitatively measure the nucleotide binding and hydrolysis by the Rag GTPases. These assays allow for a thorough investigation of this unique heterodimeric GTPase, and they could be applicable to investigations of other noncanonical GTPases.