Mitochondrial phosphopantetheinylation is required for oxidative metabolism.

4'-Phosphopantetheinyl (4'PP) groups are essential co-factors added to target proteins by phosphopantetheinyl transferase (PPTase) enzymes. Although mitochondrial 4'PP-modified proteins have been described for decades, a mitochondrially-localized PPTase has never been found in mammals. We discovered that the cytoplasmic PPTase aminoadipate semialdehyde dehydrogenase phosphopantetheinyl transferase (AASDHPPT) is required for mitochondrial respiration and oxidative metabolism. Loss of AASDHPPT results in failed 4'PP modification of the mitochondrial acyl carrier protein and blunted activity of the mitochondrial fatty acid synthesis (mtFAS) pathway. We found that in addition to its cytoplasmic localization, AASDHPPT localizes to the mitochondrial matrix via an N-terminal mitochondrial targeting sequence contained within the first 20 amino acids of the protein. Our data show that this novel mitochondrial localization of AASDHPPT is required to support mtFAS activity and oxidative metabolism. We further identify five variants of uncertain significance in AASDHPPT that are likely pathogenic in humans due to loss of mtFAS activity.