MITOL-dependent ubiquitylation negatively regulates the entry of PolγA into mitochondria

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.

Protocol to detect in vitro and in cell ubiquitylation of mitochondrial DNA polymerase gamma by mitochondrial E3 ligase MITOL

“…(Bottom panel) Equal amounts of substrates used in each condition was determined by carrying out westerns with anti-PolγA antibody. This figure is reproduced from ( Hussain et al., 2021 ) where it was presented as Figure S3A. …”

“…It is essential to prepare the wildtype versions of active recombinant E3 ligase MITOL (also known as MARCH5 or RNF153) and the target protein Polymerase gamma A (PolγA) for in vitro ubiquitylation. A catalytically-dead, recombinant version of the MITOL (called MITOL CD), detailed in ( Hussain et al., 2021 ), needs to be generated to understand the E3 ligase specificity of the ubiquitylation assay. As detailed in ( Hussain et al., 2021 ), wildtype PolγA is ubiquitylated by MITOL at position Lysine 1060 in the protein sequence.…”

“…A catalytically-dead, recombinant version of the MITOL (called MITOL CD), detailed in ( Hussain et al., 2021 ), needs to be generated to understand the E3 ligase specificity of the ubiquitylation assay. As detailed in ( Hussain et al., 2021 ), wildtype PolγA is ubiquitylated by MITOL at position Lysine 1060 in the protein sequence. Hence a recombinant PolγA mutant where Lysine 1060 is changed to arginine (PolγA K1060R) also needs to be generated to prove that MITOL ubiquitylates PolγA specifically at Lysine 1060.…”

“…Thus, we provide a detailed, reliable, highly reproducible protocol for detecting ubiquitylation of PolγA by MITOL, both in cells and in vitro . For complete details on the use and execution of this protocol, please refer to Hussain et al. (2021) .…”

“…For complete details on the use and execution of this protocol, please refer to Hussain et al. (2021) .…”