Inactivity of Peptidase ClpP Causes Primary Accumulation of Mitochondrial Disaggregase ClpX With Its Interactors at the Nucleoid and RNA Granule

Biallelic pathogenic variants in CLPP, encoding mitochondrial matrix peptidase ClpP cause a rare autosomal recessive condition, Perrault syndrome type 3 (PRLTS3). It is characterized by primary ovarian insufficiency and early sensorineural hearing loss, often associated with progressive neurological deficits. Mouse models showed that accumulations of (i) its main protein interactor, the substrate-selecting AAA+ ATPase ClpX, (ii) mitoribosomes, and (iii) mtDNA nucleoids are main cellular consequences of ClpP absence. However, the sequence of these events and their validity in human remain unclear. Here, we studied global proteome profiles to define ClpP substrates among mitochondrial ClpX interactors, which accumulated consistently in ClpP-null mouse embryonal fibroblasts and brain. Validation work included novel ClpP-mutant patient fibroblast proteomics. ClpX co-accumulated in mitochondria with POLDIP2 as nucleoid component, LRPPRC as mitochondrial poly(A) mRNA granule element, GFM1 (in mouse also GRSF1) as tRNA processing factors. Only in mouse, accumulated ClpX, GFM1 and GRSF1 appeared in nuclear fractions. Mitoribosomal accumulation was minor. Consistent accumulations in murine and human fibroblasts also affected multimerizing factors not known as ClpX interactors, namely OAT, ASS1, ACADVL, STOM, PRDX3, PC, MUT, ALDH2, PMPCB, UQCRC2 and ACADSB, but the impact on downstream metabolites was marginal. Our data demonstrate the primary impact of ClpXP on the assembly of proteins with nucleic acids, and show nucleoid enlargement in human as a key consequence.