Mitophagy

Mitophagy is an important mitochondrial quality control mechanism that eliminates damaged mitochondria. In mammals, the mitophagy pathway involves PTEN-induced putative protein kinase 1 (PINK1) and the E3 ubiquitin ligase Parkin. Learn the role of PINK1, Parkin, and the autophagy machinery in the mitophagy pathway with our interactive pathways and identify key reagents to further your research by reading the pop ups and product tables that include abcam's full product offering.

The mitophagy pathway needs to distinguish between damaged (with depolarized mitochondrial membrane potential) and healthy mitochondria. These are distinguished by the accumulation of PINK1 following mitochondrial membrane depolarization of damaged mitochondria.

In healthy mitochondria, newly synthesized PINK1 in the cytosol is imported and inserted into the mitochondrial inner membrane (IM). Then PINK1 is cleaved in its putative transmembrane domain by PARL (rhomboid-like protein) to generate the 52 kDa form of PINK1. This processed form of PINK1 is rapidly removed by a proteasome-dependent pathway, likely after its release into the cytosol from the mitochondrial intermembrane space proteins (IMS).

In unhealthy mitochondria, the inner mitochondrial membrane becomes depolarized. Upon depolarization of the mitochondrial membrane potential, the IM insertion and subsequent processing of PINK1 by PARL may be inhibited, leading to full-length PINK1 accumulating in the mitochondrial OM, probably facing the cytosol. The accumulation of PINK1 with kinase activity is sufficient for Parkin recruitment to the mitochondrial surface.

The mitophagy pathway includes the following main steps:

1. PINK1 stabilization: Under mitochondrial stress/depolarisation, full-length PINK1 is stabilized at the outer mitochondrial membrane as PINK1 import is blocked (via TOM and TIM complexes). PINK1 is subsequently cleaved by the inner membrane protease PARL.
2. PINK1 activation: When PINK1 stabilizes at the surface of the mitochondria, it is activated by auto-phosphorylation at Ser228.
3. Ubiquitin phosphorylation: Activated PINK1 phosphorylates ubiquitin at Ser65 on outer mitochondrial membrane substrates.
4. Parkin recruitment: Following phosphorylation at Ser65 by PINK1, ubiquitin binds Parkin with high affinity (cytosolic E3 ubiquitin ligase) from the cytosol to the mitochondria.
5. Parkin phosphorylation: Once at the mitochondria, Parkin is activated by both its phosphorylation at Ser65 by PINK1 and by binding to phosphorylated ubiquitin (also phosphorylated at Ser65 by PINK1)
6. Parkin-dependent substrate ubiquitination: Once active, Parkin ubiquitinates numerous substrates at the outer mitochondrial membrane. More ubiquitin chains mean more substrate for PINK1, resulting in more phosho-ubiquitin and increased Parkin recruitment in a feed-forward amplification loop.
7. Recruitment of autophagy receptors: Ubiquitin molecules at the surface of the mitochondria act as “eat me signals”, leading to the recruitment of both the ubiquitin-proteasome system (UPS) machinery and the autophagy machinery. Autophagy receptors then bridge the ubiquitinated mitochondria to the LC3-positive autophagosome. Phosphorylation of autophagy receptors by TBK1 increases interaction between LC3 and ubiquitin.
8. Autophagosome: The ubiquitinated mitochondria is engulfed by the autophagosome.
9. Fusion with lysosome: Autophagosome fuses with the lysosome allowing for the degradation of the autophagosome contents by the lysosome hydrolases.