MITOL-dependent ubiquitylation negatively regulates the entry of Pol[gamma]A into mitochondria

Mutations in mitochondrial replicative polymerase Pol[gamma]A lead to progressive external ophthalmoplegia (PEO). While Pol[gamma]A is the known central player in mitochondrial DNA (mtDNA) replication, it is unknown whether a regulatory process exists on the mitochondrial outer membrane which controlled its entry into the mitochondria. We now demonstrate that Pol[gamma]A is ubiquitylated by mitochondrial E3 ligase, MITOL (or MARCH5, RNF153). Ubiquitylation in wild-type (WT) Pol[gamma]A occurs at Lysine 1060 residue via K6 linkage. Ubiquitylation of Pol[gamma]A negatively regulates its binding to Tom20 and thereby its mitochondrial entry. While screening different PEO patients for mitochondrial entry, we found that a subset of the Pol[gamma]A mutants is hyperubiquitylated by MITOL and interact less with Tom20. These Pol[gamma]A variants cannot enter into mitochondria, instead becomes enriched in the insoluble fraction and undergo enhanced degradation. Hence, mtDNA replication, as observed via BrdU incorporation into the mtDNA, was compromised in these PEO mutants. However, by manipulating their ubiquitylation status by 2 independent techniques, these PEO mutants were reactivated, which allowed the incorporation of BrdU into mtDNA. Thus, regulated entry of non-ubiquitylated Pol[gamma]A may have beneficial consequences for certain PEO patients.