Identification of substrates of the human mitochondrial ClpXP protease and its implications for Perrault syndrome

The ClpXP ATP-dependent unfoldase-protease complex plays important roles in maintaining protein homeostasis in the mitochondria. Pathogenic variants of CLPP have been identified in patients with Perrault syndrome type 3 (PRLTS3), a rare recessive mitochondrial disease characterized by sensorineural hearing loss, ovarian dysfunction, and neurological abnormalities. However, it remains unclear how these ClpP variants cause this disease. Here we identify three cellular substrates of ClpXP using proteomic analysis of PRLTS3 patient-derived fibroblasts, with follow up studies using induced pluripotent stem cells (iPSCs) and iPSC-derived neural progenitor cells, alongside in vitro biochemical assays. These substrates are: CHCHD2, a bi-organellar regulator of oxidative phosphorylation and mitochondrial stress responses; ALAS1, which catalyzes the rate-limiting step in heme biosynthesis; and TFAM, an essential mitochondrial transcription factor. Efficient degradation of these substrates by ClpXP requires the adaptor protein PDIP38. We propose that dysregulated cellular levels of these substrates might contribute to the etiology of PRLTS3. The ClpXP ATP-dependent unfoldase-protease complex plays important roles in maintaining protein homeostasis in the mitochondria. Here, the authors identify three substrates of ClpXP that are dysregulated in PRLTS3 patients and show that degradation of these substrates requires the adaptor PDIP38.