The vast majority of mitochondrial matrix proteins are nuclear-encoded, synthesised in the cytosol and directed to the organelle via an N-terminal presequence. Processing of the presequence occurs in the matrix, generally in a single step mediated by matrix processing peptidase (MPP). Recently, a novel processing exopeptidase, intermediate cleaving peptidase (Icp55), was identified in yeast¹. Icp55 cleaves a single N-terminal amino acid from intermediates generated by MPP¹. Loss of Icp55 function generates a subset of immature matrix proteins (52 identified) that expose an N-terminal residue resembling a degradation signal (or N-degron)^1,2. Importantly, these immature matrix proteins are unstable in yeast mitochondria suggesting the existence of an N-end rule-like pathway of protein degradation in mitochondria^1,3. Mutations in the human mitochondrial homolog of Icp55, aminopeptidase P isoform 3 (APP3m) are associated with diseases such as nephronophthisis-like nephropathy and hearing loss⁴. However, its role in presequence processing, including its substrate repertoire and specificity currently remain unknown. To determine if APP3m is a functional homolog of Icp55, we have used an affinity chromatography based approach to isolate its substrates. We have also investigated the processing and turnover of one such candidate substrate in vitro, and determined that it is selectively degraded via a non-canonical N-end rule pathway by a matrix AAA+ protease. We speculate that loss of APP3m function results in the selective and premature degradation of one or more key matrix proteins resulting in mitochondrial dysfunction.