Mitochondrial Raf1 regulates glutamine catabolism.

Raf kinases are central to mitogenic signaling and cancer, yet the full complement of functionally important Raf-proximal proteins across subcellular compartments remains undefined. Here, proximity-dependent biotinylation (BioID) of Raf1 in Raf1-dependent cancer cells recovered proteins localized to the mitochondrial matrix. Mitochondrial purification and super-resolution microscopy confirmed that a pool of Raf1 resides within mitochondria. There, Raf1 associated with glutaminase (GLS) across diverse human cancers and enabled glutaminolysis, a major source of biosynthetic precursors in tumor cells. These effects required Raf1 kinase activity but were independent of canonical MAP kinase pathway signaling, and matrix-targeted kinase-dead Raf1 impaired both glutaminolysis and in vivo tumorigenesis. Raf1 therefore acts inside mitochondria, where it engages GLS to drive glutamine catabolism and support tumor growth, revealing a non-canonical, metabolic arm of Raf signaling.