The intramembrane protease PARL acts as a crucial mitochondrial safeguard by cleaving the mitophagy regulators PINK1 and PGAM5. Depending on the stress level, PGAM5 can either stimulate cell survival or cell death. In contrast to PINK1, which is constantly cleaved in healthy mitochondria and only active when the inner mitochondrial membrane is depolarized, PGAM5 processing is inversely regulated. However, determinants of PGAM5 that indicate it as a conditional substrate for PARL have not been rigorously investigated, and it is unclear how uncoupling the mitochondrial membrane potential affects its processing compared to that of PINK1. Here, we show that several polar transmembrane residues in PGAM5 distant from the cleavage site serve as determinants for its PARL-catalyzed cleavage. Our NMR analysis indicates that a short N-terminal amphipathic helix, followed by a kink and a C-terminal transmembrane helix harboring the scissile peptide bond are key for a productive interaction with PARL. Furthermore, we also show that PGAM5 is stably inserted into the inner mitochondrial membrane until uncoupling the membrane potential triggers its disassembly into monomers, which are then cleaved by PARL. In conclusion, we propose a model in which PGAM5 is slowly processed by PARL-catalyzed cleavage that is influenced by multiple hierarchical substrate features, including a membrane potential–dependent oligomeric switch.

膜内蛋白酶 PARL 通过切割线粒体自噬调节因子 PINK1 和 PGAM5 起到至关重要的线粒体保护作用。根据压力水平，PGAM5 可以刺激细胞存活或细胞死亡。与 PINK1 相比，PINK1 在健康的线粒体中不断被切割，并且仅在线粒体内膜去极化时才活跃，而 PGAM5 的加工受到反向调节。然而，表明它作为 PARL 条件底物的 PGAM5 的决定因素尚未得到严格研究，与 PINK1 相比，线粒体膜电位的解偶联如何影响其加工尚不清楚。在这里，我们展示了远离切割位点的 PGAM5 中的几个极性跨膜残基作为其 PARL 催化切割的决定因素。我们的核磁共振分析表明，一个短的 N 端两亲螺旋，然后是一个扭结和一个含有易断裂肽键的 C 端跨膜螺旋，是与 PARL 产生有效相互作用的关键。此外，我们还表明 PGAM5 稳定地插入线粒体内膜，直到解偶联膜电位触发其分解成单体，然后被 PARL 切割。总之，我们提出了一个模型，其中 PGAM5 由受多个分层底物特征影响的 PRL 催化裂解缓慢加工，包括膜电位依赖性寡聚开关。我们还表明 PGAM5 稳定地插入线粒体内膜，直到解偶联膜电位触发其分解成单体，然后被 PARL 切割。总之，我们提出了一个模型，其中 PGAM5 由受多个分层底物特征影响的 PRL 催化裂解缓慢加工，包括膜电位依赖性寡聚开关。我们还表明 PGAM5 稳定地插入线粒体内膜，直到解偶联膜电位触发其分解成单体，然后被 PARL 切割。总之，我们提出了一个模型，其中 PGAM5 由受多个分层底物特征影响的 PRL 催化裂解缓慢加工，包括膜电位依赖性寡聚开关。