Architecture of the human KICSTOR complex and its role in mTORC1 regulation

The mechanistic target of rapamycin complex 1 (mTORC1) is aberrantly activated in cancer and neurological disorders. KICSTOR, composed of KPTN, ITFG2, C12orf66, and SZT2, is required for lysosomal recruitment of GATOR1, a RagA GTPase-activating protein that inactivates mTORC1. Nevertheless, how KICSTOR localizes to lysosomes and contributes to mTORC1 regulation remains unclear. Here, we used single-particle cryo-electron microscopy, AlphaFold predictions, molecular dynamics simulations, and biochemical and cellular assays to investigate the structure and function of human KICSTOR. The largest subunit, SZT2, contains 12 repeats of a sandwich motif, termed the SZT2 repeat (SR). SR9 and SR10 are separated by an extended helix and bind KPTN and ITFG2, respectively, whereas SR12 engages C12orf66. C12orf66 contains a positively charged surface and a protruding loop that anchor KICSTOR to the negatively charged lysosomal membrane. We further show that WDR59 of GATOR2 directly interacts with KPTN in KICSTOR and GATOR1. These results uncover the architecture and lysosomal targeting mechanism of KICSTOR, offering a structural foundation for understanding mTORC1 regulation.