Abstract
Mutations in mitochondrial replicative polymerase PolγA lead to progressive external ophthalmoplegia (PEO). While PolγA 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 PolγA is ubiquitylated by mitochondrial E3 ligase, MITOL (or MARCH5, RNF153). Ubiquitylation in wild-type (WT) PolγA occurs at Lysine 1060 residue via K6 linkage. Ubiquitylation of PolγA 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 PolγA mutants is hyperubiquitylated by MITOL and interact less with Tom20. These PolγA 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 PolγA may have beneficial consequences for certain PEO patients.
Highlights
The central factor mediating mitochondrial DNA replication is the sole catalytic subunit of mtDNA polymerase γ (PolγA)
Numerical values for all graphs can be found in S1 Data
MITOL acts as a component in the mitochondrial quality control at the outer mitochondrial membrane (OMM) by ubiquitylating and degrading diverse toxicity inducing proteins like mutant SOD1, mutant short chain acyl CoA dehydrogenase, mutant MITOL itself and polyglutamine expanded protein, and mitochondrial antiviral signaling (MAVS) aggregates [20,21,22,23]
Summary
The central factor mediating mitochondrial DNA (mtDNA) replication is the sole catalytic subunit of mtDNA polymerase γ (PolγA). Together with the 2 identical subunits of the processivity factor (PolγB), PolγA forms the functional mtDNA polymerase (PolγA/B2) [1]. Mutations in POLG, the gene that codes for PolγA, are associated with mitochondrial disorders like progressive external ophthalmoplegia (PEO), Alpers–Huttenlocher syndrome (AHS), myocerebrohepatopathy spectrum (MCHS) disorders, myoclonic epilepsy myopathy sensory ataxia (MEMSA), and ataxia neuropathy spectrum (ANS). While the mutations in PolG are spread over the entire gene body, their frequency is more in the polymerase domain. Most of the common PolγA mutations (like A467T, W748S, and Y955C) have deficiency in catalytic binding and/or polymerase activity as demonstrated by in vitro assays [2,3,4].
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