ABHD4-2: a cardiolipin-selective phospholipase of the outer mitochondrial membrane

Cardiolipin (CL) is a unique dimeric phospholipid essential for mitochondrial integrity and stress signaling. While most CL is present in the inner mitochondrial membranes (IMM), CL can be exposed to the outer mitochondrial membrane (OMM) under specific physiological and pathological conditions; however, the mechanisms regulating its metabolism at the OMM are poorly defined. Based on its striking structural similarity to Cld1p, a yeast CL hydrolase, we hypothesized that α/β-hydrolase domain-containing protein 4 (ABHD4) functions as a mammalian CL hydrolase. We identified two isoforms of mouse ABHD4 arising from alternative splicing: ABHD4-1 localizes to lipid droplets, whereas ABHD4-2 selectively targets mitochondria and is enriched in oxidative tissues. We demonstrate that ABHD4 selectively hydrolyzes CL in vitro, producing monolysocardiolipin (MLCL) and dilysocardiolipin (DLCL). Site-directed mutagenesis identified catalytic serine residues as essential for enzymatic activity in ABHD4 and its Drosophila homolog, Pummelig. In contrast to Cld1p, which resides in the IMM, topological analyses indicate that ABHD4-2 and Pummelig-2 localize to the cytosolic face of the OMM, a conserved orientation that suggests a function distinct from classic acyl chain remodeling of CL. In brown adipocytes, ABHD4-2 overexpression reduces mitochondrial membrane potential in an activity-dependent manner. In silico analyses further reveal conservation of the catalytic triad across yeast, insect, and mammalian orthologs, supporting an evolutionarily conserved role for this enzyme family in CL metabolism. Together, these findings identify ABHD4-2 as an OMM-localized phospholipase with preferential CL hydrolase activity and define an isoform-specific pathway linking CL metabolism to mitochondrial stress responses.