mTORC1 is a central hub that integrates environmental cues, such as cellular stresses and nutrient availability to modulate metabolism and cellular responses. Recently, SLC38A9, a lysosomal amino acid transporter, emerged as a sensor for luminal arginine and as an activator of mTORC1. The amino acid-mediated activation of mTORC1 is regulated by the N-terminal domain of SLC38A9. Here, we determined the crystal structure of zebrafish SLC38A9 (drSLC38A9) and found the N-terminal fragment inserted deep within the transporter, bound in the substrate-binding pocket where normally arginine would bind. This represents a significant conformational change of the N-terminal domain (N-plug) when compared with our recent arginine-bound structure of drSLC38A9. We propose a ball-and-chain model for mTORC1 activation, where N-plug insertion and Rag GTPase binding with SLC38A9 is regulated by luminal arginine levels. This work provides important insights into nutrient sensing by SLC38A9 to activate the mTORC1 pathways in response to dietary amino acids.

The bedrock of drug discovery and a key tool for understanding cellular function and drug mechanisms of action is the structure determination of chemical compounds, peptides, and proteins. The development of new structure characterization tools, particularly those that fill critical gaps in existing methods, presents important steps forward for structural biology and drug discovery. The emergence of microcrystal electron diffraction (MicroED) expands the application of cryo–electron microscopy to include samples ranging from small molecules and membrane proteins to even large protein complexes using crystals that are one-billionth the size of those required for X-ray crystallography. This review outlines the conception, achievements, and exciting future trajectories for MicroED, an important addition to the existing biophysical toolkit.

Mfsd2a is the transporter for docosahexaenoic acid (DHA), an omega-3 fatty acid, across the blood brain barrier (BBB). Defects in Mfsd2a are linked to ailments from behavioral and motor dysfunctions to microcephaly. Mfsd2a transports long-chain unsaturated fatty-acids, including DHA and α-linolenic acid (ALA), that are attached to the zwitterionic lysophosphatidylcholine (LPC) headgroup. Even with the recently determined structures of Mfsd2a, the molecular details of how this transporter performs the energetically unfavorable task of translocating and flipping lysolipids across the lipid bilayer remains unclear. Here, we report five single-particle cryo-EM structures of Danio rerio Mfsd2a (drMfsd2a): in the inward-open conformation in the ligand-free state and displaying lipid-like densities modeled as ALA-LPC at four distinct positions. These Mfsd2a snapshots detail the flipping mechanism for lipid-LPC from outer to inner membrane leaflet and release for membrane integration on the cytoplasmic side. These results also map Mfsd2a mutants that disrupt lipid-LPC transport and are associated with disease.