The mTOR Complex 1 (mTORC1) pathway promotes cell growth in response to a diverse set of cues, including growth factors as well as energy and amino acid levels. Amino acids signal through the Rag GTPases to promote the translocation of mTORC1 to the lysosomal surface, its site of activation. The four mammalian Rag proteins form obligate heterodimers consisting of RagA or RagB bound to RagC or RagD. A key upstream component of the Rag GTPases is Ragulator, a trimeric complex that tethers them to the lysosome and also interacts with the v-ATPase, which is necessary for amino acid sensing by mTORC1. Amino acids stimulate the binding of GTP to RagB, a critical step in the sensing mechanism, but the factors that regulate Rag nucleotide loading are unknown. Here, we identify the proteins encoded by the HBXIP and C7orf59 genes as novel Ragulator components that are required for mTORC1 activation by amino acids. The pentameric Ragulator has nucleotide exchange activity towards RagA and RagB and interacts with the Rag heterodimers in an amino acid- and v-ATPase-dependent fashion. Thus, we provide mechanistic insight into how mTORC1 senses amino acids by revealing Ragulator to be a scaffold with guanine nucleotide exchange factor (GEF) activity for the Rag GTPases.

Interaction between different cell types is essential for multiple biological processes, including immunity, embryonic development, and neuronal signaling. While the dynamics of cell-cell interactions can be monitored in vivo by intravital microscopy19, this approach provides no information on the receptors and ligands involved, and does not allow isolation of interacting cells for downstream analysis. Here, we present a complementary approach that uses bacterial sortase labeling across immune synapses to identify receptor-ligand interactions between cells within living animals, generating a signal that can be readily detected by flow cytometry. We call this approach to labeling “kiss-and-run” interactions between immune cells Labeling Immune Partnerships by SorTagging Intercellular Contacts (LIPSTIC). Using LIPSTIC, we show that interactions between dendritic cells (DCs) and CD4+ T cells during T cell priming in vivo occur in two distinct modalities: an early, cognate stage when CD40-CD40L interactions occur specifically between T cells and antigen-loaded DCs, and a later, non-cognate stage when these interactions no longer require T cell receptor (TCR) engagement. Thus, LIPSTIC allows direct measurement of dynamic cell-cell interactions both in vitro and in vivo. Given its flexibility for use with different receptor ligand pairs and a range of detectable labels, we expect that this approach will be of use to any field of biology interested in quantifying intercellular communication.