The mammalian homologue of ClpP localizes to the mitochondrial matrix, with a tendency to associate with the inner mitochondrial membrane; mitochondrial ClpP forms two heptameric rings.

Human CLPX encodes a 633-aa precursor with an N-terminal mitochondrial transit peptide; the tagged protein is imported into mitochondria and contains an AAA+ domain and a C4 zinc-finger motif. Expression is highest in skeletal muscle and heart.

hClpX forms a hexameric ring and hClpP a heptameric ring; together with ATP they form a symmetry-mismatched ClpXP complex with ATP-dependent proteolytic and peptidase activity. Substrate selection depends solely on the Clp ATPase (CLPX).

The N-terminal peptides of CLPP line the axial channel, positioned to interact with unfolded substrates translocated by the associated CLPX chaperone; the CLPX–CLPP interaction is essential for ClpX-mediated protein and peptide degradation.

In the presence of ATP, hClpX binds hClpP and allosterically drives assembly of the active tetradecamer, conferring protease activity and greatly increased peptidase activity; CLPX thus regulates appearance of CLPP peptidase activity.

CLPX is among the chaperones/metabolic proteins identified in mitochondrial DNA nucleoid preparations, consistent with a nucleoid-associated localization.

A Walker B mutant (E359A) of human CLPX lacks ATPase activity but still mediates casein degradation by hCLPP; most model substrates are recognized by the N-terminal domain, and human CLPXP is deregulated by ADEPs.

Mitochondrial ClpX directly activates ALA synthase (ALAS) by accelerating ATP-dependent incorporation of the pyridoxal phosphate cofactor into the apoenzyme, using its central polypeptide-translocating (unfoldase) activity; this activity is conserved from yeast to mammals and is required for efficient erythropoiesis.

A dominant p.Gly298Asp mutation in the CLPX ATPase active site inactivates ATPase activity; mutant/WT coassembly yields reduced-activity enzyme that increases ALAS stability and ALA/PPIX levels, causing erythropoietic protoporphyria (EPP2). CLPX both activates and promotes turnover of ALAS.

CLPX is identified as part of the high-confidence human mitochondrial proteome.