New Players in the Mitophagy Game

Tue, 03/03/2020 - 11:03

Adenine nucleotide translocase 1 or ADP/ATP translocase (ANT) is expressed in the inner mitochondrial membrane where it exchanges free ATP and ADP from and into the mitochondrial matrix, respectively.

By Christina Towers, PhD

Mitochondrial turn over via the lysosome, otherwise known as mitophagy, involves engulfment of mitochondria into double membrane autophagosomes and subsequent fusion with lysosomes. Much is already known about this process including the canonical and non-canonical mechanisms of action, the critical machinery involved, as well as the cargo receptors that facilitate selective degradation1. Nonetheless, Hoshino et al    sought to identify novel regulators of mitophagy by performing a series of elegant genome-wide screens utilizing mitophagy specific flow cytometry assays2.

Robust Mitophagy Genetic Screen

They used flow cytometry with 4 different systems including (1) mitotracker labeled mitochondria, (2) GFP targeted to the outer mitochondrial membrane with expression of OMP25-GFP, (3) GFP targeted to the inner mitochondrial membrane with COX8-GFP, and (4) the pH sensitive probe called Keima targeted to the inner mitochondrial membrane.  In wild type cells, mitochondrial toxins will cause a decrease in total mitotracker, OMP25-GFP and Cox8-GFP expression as well as a decrease in the green/red ratio of mito-targeted Keima. After infection with viral particles carrying guide RNAs targeting over 20,000 genes, they collected the cell population that did not show a robust decrease with any of the four assays after treatment with mitochondrial toxins. They assessed the enriched guide RNAs to identify the genes that when knocked out result in the inhibition of mitophagy induction. 

PINK1 or PTEN-induced kinase 1 is a mitochondrial serine/threonine kinase which participates in mitochondrial quality control through the induction of mitophagy. Immunocytochemical analysis of HeLa cells with PINK1 Antibody (BC100-494). HeLa cells in culture were treated with 1μM valinomycin (24h). (Green) HeLa cells were stained with 20μg/mL Rabbit polyclonal PINK antibody (BC100-494) and Alexa-Fluor488 anti-rabbit secondary antibody. (Red) Tubulin was stained with anti-tubulin antibody and anti-mouse DyLight 550 secondary antibody. (Blue) Nuclei was counterstained with DAPI. Note: mitochondria staining might not be easily observed without treatment with valinomycin or CCCP.

By comparing 4 different assays to evaluate different aspects of mitochondrial turnover, combined with two different mitochondrial stressors, this screen used 7 different conditions to perform an extremely rigorous and robust analysis. Importantly, some of the top hits confirmed proteins that were already known to be essential for mitophagy including PINK1. One interesting finding was that core autophagy genes thought to be critical for mitophagy were only identified with assays that specifically monitored the mitochondrial matrix. Interestingly, autophagy genes were not important for turnover of the outer mitochondrial membrane. While there were other differences noted between the assays and the drugs used, the authors chose to focus on a novel hit that was shared across all 7 screens, adenosine nucleotide translocator (ANT).

Dissecting the Role of ANT in Mitophagy

Prior to these findings, ANT was not known to play a role in mitophagy and so the authors go on to tease apart the detailed mechanisms of regulation. In a series of experiments with exogenous WT or mutant forms of ANT rescued back into ANT KO cells, they discovered that the ATP binding domain that is necessary for ADP/ATP exchange was dispensable for ANT-mediated mitophagy induction. Instead, they found that ANT interacts with TIM44 and TIM23 to regulate PINK1 stabilization and Parkin-mediated mitophagy.

Importantly, they found that ANT also regulates mitophagy in vivo in mice as well as in people. In a patient harboring a homozygous loss of function mutation in ANT1, electron micrographs of an endomyocardial biopsy revealed severe mitochondrial abnormalities suggesting defective mitochondrial turnover. Together these studies utilized a robust, genome-wide screen to identify a previously unknown regulator of mitophagy, highlighting that there is still much that is unknown about this selective degradation process.  

Learn more about selective autophagy

Christina TowersChristina Towers, PhD   
University of Colorado (AMC)
Dr. Towers studies the roles of autophagy, apoptosis and cell death in cancer.

  1. Pickles, S., Vigié, P., & Youle, R. J. (2018). Mitophagy and Quality Control Mechanisms in Mitochondrial Maintenance. Current Biology.
  2. Hoshino, A., Wang, W. jia, Wada, S., McDermott-Roe, C., Evans, C. S., Gosis, B., … Arany, Z. (2019). The ADP/ATP translocase drives mitophagy independent of nucleotide exchange. Nature.

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