Cholesterol-dependent lysosomal docking of mTORC1 mediated by TM6SF1

The Transmembrane 6 Superfamily (TM6SF) comprises two members: TM6SF1, a ubiquitously expressed lysosomal membrane protein of unknown function, and TM6SF2, an endoplasmic reticulum protein required for lipidation of Apolipoprotein B-containing lipoproteins. Here, we identify TM6SF1 as a cholesterol-bound lysosomal docking factor for mTORC1. Loss of TM6SF1 disrupts spatial organization of the lysosomal mTORC1 machinery and results in constitutive activation of Transcription Factor EB (TFEB) without altering lysosomal pH or interfering with cholesterol trafficking. Using cryo-electron microscopy, we determined the structure of human TM6SF1 at 2.9-[A] resolution, revealing a cholesterol-bound polytopic homodimer. Biochemical analyses show that TM6SF1 directly engages LAMTOR1 to stabilize the Ragulator complex, and that disruption of cholesterol binding to TM6SF1 impairs this interaction, leading to mTORC1 mislocalization and sustained TFEB activation. These findings establish cholesterol-bound TM6SF1 as a structural determinant of mTORC1 docking, uncovering a direct mechanistic link between lysosomal cholesterol and growth control.