HUWE1 targets mitochondria via RMC1 to promote neurodevelopment.
The HECT-type E3 ubiquitin ligase HUWE1 is a critical regulator of protein homeostasis, genome stability, and neurodevelopment. Variants in HUWE1 are linked to X-linked intellectual disability (XLID), yet a significant number of HUWE1 variants are noncatalytic, leaving their disease mechanisms unresolved. Here, we identify an AMBRA1-RMC1-HUWE1 signaling axis that directs HUWE1-mediated ubiquitination to mitochondria to regulate neural development. In zebrafish, noncatalytic HUWE1 variants found in XLID patients (M375I, G660R, and H669Q) recapitulate key neurodevelopmental deficits, including impaired brain growth and motor neuron defects. Global ubiquitin profiling reveals that these variants selectively disrupt the ubiquitination of mitochondrial proteins without affecting nuclear substrates. Mechanistically, AMBRA1 acts as a mitochondrial scaffold that recruits RMC1, enabling HUWE1 localization to mitochondria through direct interaction with RMC1. Disruption of this recruitment by disease-associated HUWE1 variants prevents mitochondrial targeting and impairs mitochondrial substrate ubiquitination. Notably, Urolithin A treatment ameliorates neurodevelopmental phenotypes in HUWE1-deficient zebrafish. Together, our findings define an AMBRA1-organized mitochondrial recruitment pathway in which RMC1 links HUWE1 to mitochondria, explain noncatalytic HUWE1-associated XLID, and suggest mitochondrial quality-control enhancement as a potential therapeutic strategy.