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

Mansoor Hussain; Shabnam Saifi; Aftab Mohammed; Sagar Sengupta

doi:10.1016/j.xpro.2022.101710

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

STAR Protoc. 2022 Dec 16;3(4):101710. doi: 10.1016/j.xpro.2022.101710. Epub 2022 Sep 21.

Authors

Mansoor Hussain¹, Shabnam Saifi¹, Aftab Mohammed¹, Sagar Sengupta²

Affiliations

¹ National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
² National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India. Electronic address: sagar@nii.ac.in.

Abstract

Mitochondrial polymerase gamma (PolγA) is the only replicative polymerase in mitochondria. To determine PolγA ubiquitylation in cells, Flag-PolγA and MITOL are overexpressed, and subsequently the immunoprecipitated Flag-PolγA is checked for ubiquitylation. Alternately, in vitro synthesized PolγA and MITOL are used to determine whether PolγA is ubiquitylated. Either anti-ubiquitin or anti-Flag antibody is used to detect the ubiquitylated product. 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).

Keywords: Cell biology; Genetics; Molecular biology; Protein biochemistry; Protein expression and purification.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

DNA Polymerase gamma / genetics
DNA Polymerase gamma / metabolism
Mitochondria* / metabolism
Ubiquitin / metabolism
Ubiquitin-Protein Ligases* / metabolism
Ubiquitination

Substances

Ubiquitin-Protein Ligases
DNA Polymerase gamma
Ubiquitin