CALHM2 is a mitochondrial protein import channel that regulates fatty acid metabolism.

For mitochondrial metabolism to occur in the matrix, multiple proteins must be imported across the two (inner and outer) mitochondrial membranes. Classically, two protein import channels, TIM/TOM, are known to perform this function, but whether other protein import channels exist is not known. Here, using super-resolution microscopy, proteomics, and electrophysiological techniques, we identify CALHM2 as the import channel for the ECHA subunit of the mitochondrial trifunctional protein (mTFP), which catalyzes β-oxidation of fatty acids in the mitochondrial matrix. We find that CALHM2 sits specifically at the inner mitochondrial and cristae membranes and is critical for membrane morphology. Depletion of CALHM2 leads to a mislocalization of ECHA outside of the mitochondria leading to severe cellular metabolic defects. These defects include cytosolic accumulation of fatty acids, depletion of tricarboxylic acid cycle enzymes and intermediates, and reduced cellular respiration. Our data identify CALHM2 as an essential protein import channel that is critical for fatty acid- and glucose-dependent aerobic metabolism.